Thymidine kinase deletion mutants of bovine herpesvirus-1, vaccines against infectious bovine rhinotracheitis containing same and methods for the production and use of same

ABSTRACT

Bovine herpesvirus type 1 (infectious bovine rhinotracheitis virus) mutants which fail to produce any functional thymidine kinase as a result of a deletion in the thymidine kinase gene. The deletion in the thymidine kinase gene attenuates the viruses so that they can be used as an active agent in a modified live-virus vaccine against infectious bovine rhinotracheitis. This invention also relates to methods for the production and use of the same.

This is a division of application Ser. No. 796,840 filed Nov. 12, 1985,now U.S. Pat. No. 4,703,011.

FIELD OF THE INVENTION

The present invention relates to bovine herpesvirus type 1 viruses whichfail to produce any functional thymidine kinase as a result of adeletion in the thymidine kinase gene, vaccines against infectiousbovine rhinotracheitis containing the same and methods for theproduction and use of the same.

BACKGROUND OF THE INVENTION I. Infectious Bovine Rhinotracheitis

Bovine herpesvirus type 1 (hereinafter "BHV-1"), more commonly known asinfectious bovine rhinotracheitis virus (hereinafter "IBRV"), has beenassociated with respiratory, reproductive, enteric, occular, centralnervous system, neonatal, mammary, and dermal infections of cattle.Evidence for the association of IBRV with diseases of the respiratorytract was first obtained in the early 1950's. It has since becomeapparent that infectious bovine rhinotracheitis (hereinafter "IBR") hasa worldwide distribution. Clinical symptoms are characterized by asudden onset of hyperthermia, anorexia, and depression. The severeinflammation of the epithelial surfaces of the respiratory membranesoften progresses to a necrotic rhinotracheitis (see: Schroeder, R.J. andMoys, M.D., J. Am. Vet. Med. Assoc. 125:471-472 (1954); McKercher, D.G.,Moulton, J.E., and Jasper, D.E., Proc. U.S. Livestock Sanit. Assoc.58:260-269 (1955); Gibbs, E.P.J. and Rweyemamu, M.M., The Vet. Bull.47:317-343 (1977); Kahrs, R.F., J. Am. Vet. Med. Assoc. 171:1055 1066(1977); Moorthy, A.R.S., Vet. Record 116:98 (1985); Ross, H.M., Vet.Record 113:217-218 (1983); Guy, J.S., Potgieter, L.N.D., McCracken, M.,and Martin W., Am. J. Vet. Res. 45:783-785 (1984); and Engels, M.,Steck, F., and Wyler, R., Arch. Virol. 67:169-174 (1981)).

In natural outbreaks of the respiratory form of the disease,conjunctivitis, manifested by a copious discharge, extensive hyperemiaand edema of the conjunctiva, is also prominent. Infectious pustularvulvovaginitis and balanoposthitis are also caused by BHV-1, and arecharacterized by hyperemia of the vulvovaginal and preputial mucousmembranes. This can lead to pustule formation and ulceration.

The spread of the disease in naturally and artificially bred cattleposes a serious problem, especially with the continued, widespread useof frozen semen. Recurrent shedding of virus from infected bulls alsoconstitutes a significant threat to the artificial insemination industryin the United States and to the worldwide distribution of bovine germplasm. The incrimination of BHV-1 as an etiologic agent of oophoritisand salpingitis with resultant infertility and sterility adds to theseriousness of the infection.

BHV-1 is widely recognized as a cause of abortion, stillbirths, andinfertility. Most naturally occurring abortions occur between the fourthand seventh months of gestation, but cattle may abort from BHV-1infections throughout gestation. Respiratory disease and conjunctivitismay or may not be observed prior to abortion.

Meningoencephalitis is another of the sequela to BHV-1 infection.Neurotropic symptoms are observed most often in calves under 6 months ofage. The rate of encephalitis may vary from an occasional animal to alarge portion of the herd.

BHV-1 infections of species other than cattle have been described (see:Fulton, R.W., Downing, M.M, and Hagstad, H.V., Am. J. Vet. Res.43:1454-1457 (1982) and Lupton, H.W., Barnes, H.J., and Reed, D.E.,Cornell Vet. 70:77-95 (1980)). Natural infections occur in swine, goats,water buffalo, wildebeests, ferrets, and mink. BHV-1 has been blamed forepizootics of vaginitis and balanitis in swine, and BHV-1 has beenisolated from stillborn and newborn pigs in herds with a history ofreproductive problems. According to serologic studies, about 11% ofswine sera from Iowa and Texas herds contain BHV-1 antibody titers.Experimental infections have been established in swine fetuses, goats,mule deer, ferrets, and rabbits (see: Joo, H.S., Dee, S.A., Molitor,T.W., and Thacker, B.J., Am. J. Vet. Med. Assoc. 45:1924 1927 (1984)).

The severity of illness resulting from BHV-1 infections depends upon thevirus strain and on the age of the animal affected. After recovery frominfection, animals may show clinical signs of recurrent disease withoutbeing reexposed to the virus. Recurrent disease without reexposureoccurs because the virus remains dormant, i.e. latent, in neurons of thesensory ganglia of its host and can be reactivated, even after longperiods (see: Homan, E.J. and Easterday, B.C., J. Infect. Dis. 146:97(1982); Homan, E.J. and Easterday, B.C., Am. J. Vet. Res. 44:309-313(1983); and Ackermann, M., Peterhans, E., and Wyler, R., Am. J. Vet.Res. 43:36-40 (1982)). Dexamethasone treatment can also provoke nasalshedding of the virus with or without clinical symptoms of active IBR.This suggests that reactivation and release from neuronal sites and,possibly, persistent infection of other tissues can occur (see: Rossi,C.R., Kiesel, G.K., and Rumph, P.F., Am. J. Vet. Res. 43:1440-1442(1982)).

II. Known IBR Vaccines

Currently, three types of IBR vaccines are being employed: (1) killedvirus vaccines, (2) subunit vaccine, and (3) modified live-virus(hereinafter "MLV") vaccines (see: U.S. Pat. Nos. 3,925,544; 4,291,019;and 3,634,587). Killed IBR vaccines are produced by treating the viruswith chemicals, such as formalin or ethanol and/or physical agents, suchas heat or ultraviolet irradiation. Subunit IBR vaccines are prepared bysolubilizing BHV-1-infected cell cultures with nonionic detergents.Early MLV vaccines were designed for parenteral administration andconsisted of virus attenuated by rapid passage in bovine cell cultures.More recently, parenterally administered MLV vaccines have beenattenuated by adaption of BHV-1 to porcine or canine cell cultures, byadaption to growth in cell culture at a low temperature (30° C), or byselection of heat-stable virus particles (56° C. for 40 minutes).Specialized types of MLV vaccines are those administered intranasally.These MLV vaccines are attenuated by serial passage in rabbit cellcultures or by treatment of BHV-1 with nitrous acid followed byselection for temperature-sensitive mutants. A temperature-sensitivevirus is one that replicates efficiently at about 32° C.-38° C., but notat about 39° C. -41° C. (see: Todd, J.D., Volenec, F.J., and Paton,I.M., J. Am. Vet. Med. Assoc. 159:1370-1374 (1971); Kahrs, R.F.,Hillman, R.B., and Todd, J.D., J. Am. Vet. Med. Assoc. 163:437-441(1973); Smith, M.W., Miller, R.B., Svoboda, I., and Lawson, K.F., Can.Vet. J. 19:63-71 (1978); Zygraich, N., Lobmann, M., Vascoboinic, E.,Berge, E., and Huygelen, C., Res. Vet. Sci. 16:328-335 (1974); and U.S.Pat. Nos. 3,907,986 and 4,132,775).

All of the currently available IBR vaccines have serious disadvantagesand have, therefore, proved unsatisfactory in commercial use. Morespecifically, although killed IBR vaccines are considered by some to besafer than MLV vaccines, i.e., they cannot establish latency and theyeliminate the problem of postvaccination shedding, they are expensive toproduce, must be administered several times, and disadvantageouslyrequire adjuvants. In addition, with their use, there is the possibilityof fatal hypersensitivity reactions and nonfatal urticaria. Further,some infectious virus particles may survive the killing process and thuscause disease. Moreover, cattle vaccinated with killed IBR vaccines canbe infected at a later time with virulent virus and can shed virulentvirus, thereby spreading infection in the herd (see: Frerichs, G.N.,Woods, S.B., Lucas,

Sands, J.J., Vet. Record 111:116-122 (1982)). In one study, a killed IBRvaccine using a 5-component adjuvant was found entirely ineffective inproducing immunity, preventing disease, and suppressing propagation andreexcretion of virulent virus (see: Msolla, P.M., Wiseman, A., Selman,I.E., Pirie, H.M., and

Allen, E.M., Vet. Record 104:535-536 (1979)). That is, followingchallenge exposure to virulent virus, vaccinated animals exhibitedclinical signs and virus excretion responses virtually identical tounvaccinated animals. Further, in marked contrast to calves whichrecovered from natural infection, these calves transmitted virulentvirus to in-contact controls. Thus, although killed IBR vaccines canprovide some protection against IBR, they are generally inferior to MLVvaccines in providing long-term protection.

Subunit vaccines are often less toxic than killed virus vaccines, andmay induce novel immunologic effects which can be of significant value.The technique for subunit vaccine preparation involves removal of capsidproteics, while leaving intact antigens that elicit protective immunity.This creates a potential for the development of serologic procedures todifferentiate vaccinated from naturally infected animals. Further,subunit vaccines may be antigenic, yet contain no live virus and, thus,cannot be transmitted to other animals, cause abortion, or establishlatency. In one study, a single dose of subunit vaccine markedlymodified challenge infection, and two doses prevented clinical diseaseand virus shedding in all vaccinated animals after challenge exposurewith 10⁶ PFU of the standard virulent BHV-1(Cooper) strain inoculum.However, the possibility that a latent infection was established couldnot be excluded (see: Lupton, H.W. and Reed, D.E., Am. J. Vet. Res.41:383-390 (1980)). When testing another IBR subunit vaccine previouslyfound to elicit a strong immune response in adult cattle, the vaccinefailed to do so in younger animals and it did not protect the animalsagainst respiratory disease (see: le Q. Darcel, C. and Jericho, K., Can.J. Comp. Med. 45:87-91 (1981)). Other disadvantages of subunit vaccinesare the high cost of purification and the requirement of severalinjections with adjuvant.

MLV IBR vaccines have the important advantage that they produce rapidprotection and activate cellmediated and humoral components of theimmune system. In the case of intranasal vaccination, localized immuneresponses that suppress later replication of virulent BHV-1 in therespiratory tract contribute significantly to protection. The localimmune responses include production of interferon and antibodies innasal secretions (see: Kucera, C.J., White, R.G., and Beckenhauer, W.H.,Am. J. Vet. Res. 39:607-610 (1978)).

Extensive utilization of MLV IBR vaccines has reduced the frequency ofoccurrence of IBR. However, none of the available MLV IBR vaccines areentirely satisfactory. More specifically, there is concern as to theirsafety, especially if the vaccine virus itself produces latency and maybe shed and transmitted to susceptible cattle. Vaccination withavailable MLV preparations is also ineffective in preventing latentinfections following exposure to virulent BHV-1. For example, in onestudy, a MLV IBR vaccine, obtained by passing BHV-1 43 times in porcinetestes cells followed by 8 passages in monolayer cultures of bovinetestes at 30° C, was used to vaccinate calves (see: Narita, M., Inui,S., Nanba, K., and Shimizu, Y., Am. J. Vet. Res. 41:1995-1999 (1980)).At 49 days after the calves were challenge-exposed to virulent BHV-1,the calves were treated with dexamethasone and latent virus infectionwas demonstrated through signs of recurrent infection.

Maximal utilization of intramuscularly (hereinafter "IM") administeredMLV IBR vaccines has been especially hampered by the hazards ofvaccine-induced abortions. That is, abortion rates as high as 60% havebeen reported after IM injection of some MLV IBR vaccines (see: Kahrs,R.F., J. Am. Vet. Med. Assoc. 171:1055-1064 (1977) and Kendrick, J.W.and Straub, O.C., Am. J. Vet. Res. 28:1269-1282 (1967)). In addition,with the MLV IBR vaccines currently in use, there is the danger ofreversion to virulence.

In a search for safer MLV IBR vaccines, specialized vaccines have beendeveloped (see: Todd, J.D., Volenec, F.J., and Paton, I.M., J. Am. Vet.Med. Assoc. 159:1370-1374 (1971); Kahrs, R.F., Hillman, R.B., and Todd,J.D., J. Am. Vet. Med. Assoc. 163: 427-441 (1973); Smith, M.W., Miller,R.B., Svoboda, I., and Lawson, K.F., Can. Vet. J. 19:63-71 (1978);Zygraich, N., Lobmann, M., Vascoboinic, E., Berge, E., and Huygelen, C.,Res. Vet. Sci. 16:328-335 (1974); and Kucera, C.J., White, R.G., andBeckenhauer, W.H., Am. J. Vet. Res. 39:607-610 (1978)). These vaccineshave been found to be immunogenic and safe for intranasal (hereinafter"IN") inoculation to pregnant cattle and can prevent abortions inpregnant cows which have been challenge-exposed to virulent BHV-1.However, they have a disadvantage in that they can only be administeredby the IN route. This is because, when administered IN, one such IBRvaccine replicates to a limited extent at the lower temperature of theupper respiratory tract. However, when administered IM, the vaccinereplicates poorly or not at all at normal body temperature (see:Zygraich, N., Lobmann, M., Vascoboinic, E., Berge, E., and Huygelen, C.,Res. Vet. Sci. 16:328-335 (1974)). On the other hand, another IBRvaccine is insufficiently attenuated for IM administration to pregnantanimals although safe when given IN (see: Todd, J.D., J. Am. Vet. Med.Assoc. 163: 427-441 (1973). Furthermore, some of the vaccine strainsproduce mild or moderate respiratory disease even after INadministration, and they do not prevent signs of IBR following fieldchallenge exposure (see: Kahrs, R.F., Hillman, R.B., and Todd, J.D., J.Am. Vet. Med. Assoc. 163:437-441 (1973)).

Accordingly, neither the IM-administered MLV IBR vaccines, which areunsafe for pregnant cows, nor the MLV IBR vaccines that must beadministered IN fits comfortably into many of the current managementpractices. That is, vaccination of large numbers of cattle by the INroute is inconvenient and potentially dangerous to animal handlers.Additionally, screening to identify pregnant animals prior toimmunization is often not desirable or cost effective. The developmentof a vaccine which can be safely administered either IN or IM instressed feedlot cattle, in breeding bulls, nd even in pregnant cowswould facilitate disease prevention, be more cost effective, and becompatible with current management regimens. The present invention wasdeveloped in order to meet these needs.

III. Attenuated Properties of Thymidine Kinase-Negative HerpesvirusMutants

Recently, a temperature-resistant, thymidine kinase-negative(hereinafter "tk⁻ ") IBR vaccine has been developed which overcomes manyof the problems that have limited the use of currently available vac 7,cines (see: Kit, S. and Qavi, H., Virol. 130:381-389 (1983) and U.S.patent application Ser. No. 516,179, filed July 21, 1983). This BHV-1mutant replicates equally well in rabbit skin and in bovine tracheal orbovine turbinate cells at either 39.1° C. or 34.5° C. In contrast,temperature-sensitive BHV-1 strains replicate only 10⁻⁴ to 10⁻⁷ as wellat 39.1° C. as at 34.5° C. In addition, this mutant lacks the ability toproduce functional thymidine kinase (hereinafter "TK") enzyme activityin infected cells as a result of a mutagen-induced mutation. These twocharacteristics, i.e. temperature resistance and tk⁻, directlycontribute to the superiority of this virus as a vaccine as discussed inmore detail below.

Temperature-sensitive viruses are a specialized type of attenuated virusand contain mutations in genes essential for virus replication. That is,they are "crippled" viruses and replicate poorly, if at all, at normalbody temperatures. Therefore, IN-administered vaccine viruses withtemperature-sensitive mutations must be restricted to the nasal mucosaand the surface epithelial cells of the upper respiratory tract, whichhas a lower temperature. Further, IN-administered vaccine virusesregularly shed more virus in nasal secretions than IM-administeredvaccine viruses, and they produce latency as readily as do regularnon-temperature-sensitive viruses. On the other hand,temperature-resistant viruses replicate efficiently at 39.1° C. and arenot attenuated through "crippling" mutations in genes required for virusreplication. Further, they provide stronger immune responses thantemperature-sensitive viruses because the resistance to highertemperatures allows them to replicate efficiently in tissues deep withinthe body and in febrile animals. Moreover, the option exists ofadministering these viruses by IM, IN, or other routes.

Herpesvirus-encoded TK enzymes are distinct from host cell TK enzymes inimmunological and biochemical properties. Herpesvirus TK enzymesfacilitate herpesvirus replication in nondividing cells. Because manyherpesviruses are neurotropic viruses normally capable of bothproductive and latent infection in nondividing neural cells, it has beenhypothesized that tk⁻ virus mutants may be less neurovirulent thanwild-type thymidine kinase-positive (hereinafter "tk⁺ ") strains.

Many recent experiments utilizing animal model systems demonstrate thatherpes simplex type 1 (hereinafter "HSV-1") tk genes are important forvirulence (see: Klein, R.J., Arch. Virol. 72:143-160 (1982)). Thesestudies have shown that tk⁻ mutants of HSV-1 have reduced pathogenicityin mice, rabbits, and guinea pigs for herpes encephalitis, herpeskeratitis, and herpes labialis. In addition, the tk⁻ HSV-1 mutants: (i)are less likely to be reactivated from latency; (ii) protect laboratoryanimals against fatal infection from virulent tk⁺ viruses; and (iii)reduce the probability of colonization of sensory ganglia bysuperinfecting virulent tk⁺ viruses. tkpseudorabies mutants of HSV-2,Herpesvirus tamarinus, and virus are also less virulent than parentaltk⁺ strains. Furthermore, restoration of the tk⁺ function by recombiningtk⁻ pseudorabies virus mutants with DNA fragments encoding the tk geneincreases the virulence for mice, whereas converting the latterrecombinant tk⁺ viruses once more tk⁻ mutants again diminishes virulencefor mice (see: Kit, S., Qavi, H., Dubbs, D.R., and Otsuka, H., J. Med.Virol. 12:25-36 (1983) and Kit, S., Kit, M., and Pirtle, E.C., Am. J.Vet. Res. 46:1359-1367 (1985)).

In guinea pigs, tk⁺ strains of HSV-2 replicate to high titers in thevagina and in the spinal cord. The guinea pigs infected intravaginallywith tk⁺ viruses exhibit severe vesiculoulcerative genital lesions,urinary retention, hind-limb paralysis, and death in about 25% to 33% ofthe animals. The onset, magnitude, and duration of vaginal virusreplication is about the same following tk HSV-2 inoculation as thatobserved after tk HSV-2 inoculation. However, guinea pigs inoculatedwith tk⁻ HSV-2 exhibit no deaths, little or no clinical illness, andonly low titers of virus are detected in spinal cord homogenate cultures(see: Stanberry, L.R., Kit, S., and Myers, M.G., J. Virol. 55:322-328(1985)). In addition, vaccination of guinea pigs with tk⁻ HSV-2 modifiesa subsequent tk⁺ HSV-2 genital infection. That is, reinfection with tk⁺HSV-2 is clinically inapparent, vaginal replication of tk⁺ HSV-2 isreduced, and ganglionic infection is prevented.

In addition to the preceding model experiments with laboratory animals,the attenuated properties of a tk⁻ deletion mutant of pseudorabies virusfor natural hosts has been demonstrated (see: U.S. Pat. No. 4,514,497and Kit, S., Kit, M. and Pirtle, E.C., Am. J. Vet. Res. 46:1359-1367(1985)).

Pilot experiments performed in 5- to 6-week-old pigs provided theinitial evidence for the safety and efficacy of a pseudorabies virus tk⁻deletion mutant. These experiments demonstrated that pigs vaccinated IMor IN with 7.5×10⁸ PFU of the pseudorabies virus tk⁻ deletion mutant andthen challenge-exposed IN with the very high dose of 6×10⁸ PFU of thehighly virulent Indiana-Funkhauser (Ind-F) strain of pseudorabies virus,had no clinical signs of illness after vaccination or after challengeexposure. Nonvaccinated pigs either died or became moribund beforeeventually recovering. In another study on a quarantined swine herd inTexas, more than 700 grower-finisher pigs, 224 nursery pigs, 128 femalesin all stages of pregnancy, 56 nonpregnant females, 7 boars, and 224piglets were immunized with no adverse reactions (see: Kit, S., Kit, M.,Lawhorn, B., and McConnell, S., ASM Publication on Proceedings of the1984 High Technology Route to Virus Vaccines, American Society forMicrobiology, Washington, D.C., pp. 82-99 (1985)). Finally, highlysusceptible calves and weanling lambs were vaccinated with over 10⁸ PFUof the pseudorabies virus tk⁻ deletion mutant, but no disease signs wereobserved.

Two pilot studies in the natural host to assess the safety and efficacyof a temperature-resistant tk⁻ mutagen-induced mutant BHV-1 vaccinevirus, i.e., BHV-1(B8-D53) (ATCC No. VR-2066) have also been completed(see: Kit, S., Qavi, H., Gaines, J.D., Billingsley, P., and McConnell,S., Arch. Virol. 86:63-84 (1985)).

In the first study, four groups of calves consisting of 19 Holsteinsteers and 1 freemartin, 3 months of age were used. Group 1 consisted of7 calves vaccinated either IN or intravenously with 2×10⁷ PFU ofBHV-1(B8-D53) and challenged IN 56 days later with either 1.3×10⁸ PFUBHV-1(Cooper , i.e., the U.S. Department of Agriculture standardchallenge BHV-1, or with 2×10⁷ PFU of BHV-1(Los Angeles), i.e., theparental strain from which BHV-1(B8-D53) was derived. Group 2 consistedof 3 calves vaccinated as above but not challenge-exposed with virulentBHV-1. Group 3 consisted of 4 calves that were not vaccinated, but werechallenge-exposed IN with either the Cooper or Los Angeles strain ofBHV-1. Group 4 consisted of 6 control calves that were neithervaccinated nor challenge-exposed. At 91 days after vaccination, calvesfrom all four groups were stressed for 5 days with an intravenousinjection of dexamethasone. On day 121 after vaccination, thedexamethasone treatment was repeated.

Vaccinated calves developed neutralizing antibodies but did not showclinical signs of IBR disease following IN challenge exposure to thevirulent BHV-1 strains. BHV-1(B8-D53) vaccination reduced themultiplication of virulent BHV-1 in the nasal mucosa, but did notcompletely prevent development of a persistent infection by thechallenge virus.

In the second study, 15 pregnant cows, seronegative for BHV-1, wereused.

The cows were randomly assigned into one of two groups: (a) 5 cows to bevaccinated IM plus 2 contact controls; and (b) 3 cows to be vaccinatedIM, 3 cows to be vaccinated intravaginally, and 2 contact controls. Onday 1, 3.0×10⁷ PFU of BHV-1 B8-D53) vaccine virus was given either by IMinoculation or by intravaginal instillation.

On day 45, the cows were segregated and randomized again for thevirulent BHV-1(Cooper) challenge exposure segment of the study. The twogroups consisted of (a) 5 IM- and 1 intravaginally vaccinated cows plus1 nonvaccinated, nonchallenge-exposed contact control cow, and (b) 3 IM-and 2 intravaginally vaccinated cows, 2 nonvaccinated, challenge-exposedcontrol cows, and 1 nonvaccinated, nonchallenge-exposed contact controlcow.

The group (b) cows (excluding the contact control) werechallenge-exposed IN on day 46, with 2.8×10⁹ PFU of virulentBHV-1(Cooper).

All of the pregnant cows remained clinically normal throughout the12-day observation period after BHV-1(B8-D53) vaccination.

After IN challenge exposure to virulent BHV-1(Cooper), replication IN ofthe challenge virus was detected, but the vaccinated cows did notdevelop any fever or clinical signs of disease. The nonvaccinatedchallenge control cows did show fever for 2-5 days and signs ofrespiratory distress, labored breathing, mucopurulent nasal discharge,and reduced food intake.

Live calves were born to all 15 of the pregnant cows used in this study.Precolostrum serum samples were obtained from calves born to 5 cows. Allof these precolostrum samples were negative for BHV-1 virus-neutralizingantibodies, showing that neither the IM- or the intravaginallyadministered vaccine virus, nor the IN-inoculated challenge virus hadinfected the fetuses. Postcolostrum samples from calves born toseropositive cows were positive for virus-neutralizing antibodies.

In conclusion, the lack of clinical disease in the pregnant cowsvaccinated with BHV-1(B8-D53) and challenge-exposed to BHV-1(Cooper) andthe absence of BHV-1 antibodies in the precolostrum sera of newborncalves demonstrated the avirulence of this vaccine virus.

IV Thvmidine Kinase-Neqative Herpesvirus Mutants

Several different types of herpesvirus mutants deficient in TK-inducingactivity are known. Many of these mutants induce the synthesis of normalsized TK polypeptides, but without any functional TK activity. Thesemutants either have changed amino acids at the active centers of theenzyme or mutations that alter polypeptide folding. Other mutants, i.e.,HSV-1(B2006), fail to induce the production of any TK polypeptide,possibly as a result of nonsense mutations at codons near the aminoterminus of the polypeptide. Still other mutants induce inactive,truncated (or shortened) polypeptides because of translation-terminating(nonsense) codons in the middle of the tk gene. This type of mutationcan be "suppressed" with "suppressor tRNAs" so that partial TK activityis restored. Yet other types of mutants induce TK enzymes with alterednucleoside substrate specificities. For example, wild-type (i.e.,non-mutant) HSV-1 viruses induce TK enzymes that efficiently catalyzethe phosphorylation of thymidine, deoxycytidine, and the nucleosideanalogs, bromovinyldeoxyuridine, and acyclovir. However, HSV-1 mutantswith altered substrate specificities induce TK activities with reduced,but significant, thymidine phosphorylating activity, yet no detectableacyclovir phosphorylating activity (or deoxycytidine orbromovinyldeoxyuridine phosphorylating activities). These mutants havechanged amino acids at the nucleoside binding sites of the enzymes.Hence, affinities for nucleoside substrates are reduced in comparisonwith that of wild-type HSV-1-encoded TKs. Finally, tk⁻ mutants withnucleotide deletions within the coding region of the tk gene fail toinduce production of any functional TK polypeptide.

tk⁻ herpesviruses with deletions in the tk gene are superior tospontaneous or mutagen-induced tk⁻ mutants (see: U.S. patent applicationSer. No. 516,197, filed July 21, 1983, and U.S. Pat. No. 4,514,497)containing only nucleotide changes in the tk gene for the followingthree reasons.

First, the herpesvirus deletion mutants have absolutely no TK-inducingactivity because: (i) the deletion of amino acid coding sequenceschanges the tertiary structure and/or substrate binding sites of theTK-enzyme in such a way that catalytic function is irrevocably lost; and(ii) those nucleotide deletions that are not divisible by 3 change thetranslational reading frame of the virus gene in coding the TKpolypeptide. In contrast, mutants containing nucleotide changes onlyfrequently exhibit partial TK-inducing activity. This is inappropriatefor a vaccine because mutants with partial TK activity are oftenvirulent (see: Gordon, Y., Gilden, D.H., Shtram, Y., Asher, Y., Tabor,E., Wellish, M., Devlin, M., Snipper, D., Hadar, J., and Becker, Y.,Arch. Virol. 76:39-49 (1982); Klein, R. J., Arch. Virol. 72:143-168 ((1982); and Tenser, R.B., Ressel, S., and Dunstan, M.E., Virol.112:328-334 (1981)).

Second, tk⁻ herpesviruses with deletions in the tk gene cannot revert totk⁺. In contrast, tk⁻ mutants containing only nucleotide changes in thetk gene can revert to tk⁺. tk⁺ revertants have restored virulence (see:Kit, S., Kit, M., and Pirtle, E.C., Am. J. Vet. Res. 46:1359-1367(1985)), and working pools of tk⁻ mutants can contain spontaneous tk⁺revertants at a frequency of 10⁻³ to 10⁻⁵. These tk⁺ revertants can thenhave a selective advantage for in vivo replication over the tk⁻ mutants.Most drug-induced tk⁻ herpesvirus mutants have the potential to revert,even though the reversion frequency may be lower than the spontaneousreversion frequency, i.e., on the order about 10⁻⁵ to 10⁻⁷ (see:Campione-Piccardo, J., Rawls, W.E., and Bacchetti, S., J. Virol. 31:286-287 (1979)).

Third, tk⁻ herpesviruses with deletions in the tk gene can bedistinguished from virulent field strains and from other vaccine strainsby their tk⁻ phenotypes, by their restriction endonuclease patterns, andby their Southern blotting molecular hybridization patterns (see: Kit,S., Kit, M., and Pirtle, E.C., Am. J. Vet. Res. 46:1359-1367 (1985)).These distinctions have practical importance. For example, if avaccinated animal develops disease, it is important to know whether thevaccine virus caused the disease or whether infection by a virulentfield strain did so.

General approaches for obtaining herpesviruses with deletions in thecoding region of the tk gene are known. These methods have been used toobtain tk⁻ deletion mutants of HSV-1, HSV-2, Herpesvirus tamarinus, andpseudorabies virus (see: Smiley, J.R., Nature 385:333-335 (1980); Post,L.E., Mackem, S., Roizman, B., Cell 24:555-565 (1981); Post, L.E. andRoizman, B., Cell 25:227-232 (1981); McDermott, M.R., Smiley, J.R.,Leslie, P., Brais, J., Rudzroga, H.E., and Bienenstock, J., J. Virol.51:747-753 (1983), Kit, S., Qavi, H., Dubbs, D.R., and Otsuka, H., J.Med. Virol. 12:25-36 (1983); U.S. Pat. No. 4,514,497 and Kit, S., Kit,M., and Pirtle, E.C., Am. J. Vet. Res. 46:1359-1367 (1985)).

However, these approaches are not directly useful in the preparation ofIBRV tk⁻ deletion mutants since there is no teaching or suggestiontherein of: (i) the identity of the IBRV DNA fragment that encodes thetk gene or for that matter its location on the IBRV genome; (ii) theapproximate boundaries of the IBRV tk gene; or (iii) its nucleotidesequence. Heretofore, it has only been known that: (i) IBRV possesses adouble-stranded, linear DNA genome of about 86 megadaltons (137 kilobasepairs (hereinafter "kb" or "Kbp"), and an overall molar guanine pluscytosine content of about 70%; (ii) the IBRV genome is composed of L andS components which can exist in two isomeric forms; and (iii) the DNAgenome of the Cooper strain of IBRV is cleaved by HindIII, BamHI, EcoRI,and HpaI restriction endonucleases to 15, 11, 7, and 7 fragments,respectively. The sizes of these fragments and their arrangement on thephysical map of IBRV DNA have also been described (see: Mayfield, J.E.,Good, P.J., Van Oor, H.J., Campbell, A.R., and Reed, D.E., J. Virol.47:259-264 (1983)).

To identify DNA fragments encoding herpesvirus tk genes, one or more ofthe following methods have previously been used: (i) molecularhybridization experiments, that is, experiments in which labeled DNAfragments from a known viral tk gene and DNA fragments from the unknownvirus are hybridized to detect homologous nucleotide sequences; (ii)biochemical transformation of mutant tk⁻ cells (e.g., mouse fibroblastLM(TK⁻)) to the tk⁺ phenotype; (iii) transfection or microinjection ofhybrid plasmids containing the putative tk gene into LM(TK⁻) cells ormouse or frog eggs and then assaying for transient expression of TKenzyme activity; and (iv) marker transfer experiments with infectiousDNA from a tk⁻ mutant virus and hybrid plasmids containing the tk gene(see: Scangos, G. and Ruddle, F.H., Gene 14:1-10 (1981); Brinster, R.L.,Chen, H.Y., Warren, R., Sarthy, A., and Palmiter, R.D., Nature 296:39-42(1982 ); Otsuka, H., Hazen, M., Kit, M., Qavi, H., and Kit, S., Virol.113:196-213 (1981); Kit, S., Qavi, H., Hazen, M., Trkula, D., andOtsuka, H., Virol. 113:452-464 (1981); Weir, J.P., Bajszar, G., andMoss, B., Proc. Nat. Acad. Sci. USA 79:1210-1214 (1982); Dubbs, D.R.,Otsuka, H., Qavi, H., and Kit, S., Virol. 126:408-411 (1983); Otsuka, -H., Qavi, H., and Kit, S., Antiviral Res. 2:301-311 (1982); McKnight,S.L. and Gavis, E.R., Nucl. Acids Res. 8:5931-5948 (1980); and Capecchi,M.R., Cell 22:479-488 (1980)).

However, in general, these methods are not applicable to theidentification of the DNA fragment which encodes the IBRV tk gene. Morespecifically, high specific activity, ³² P-labeled probes made bynick-translating cloned HSV, pseudorabies virus, and Herpesirustamarinus tk genes do not hybridize to IBRV DNA fragments. Thus, thetechnique of molecular hybridization is not applicable to IBRV. That is,there is substantially no homology between the tk genes of the variousherpesvirus species, i.e. the nucleotide sequences of the tk genes andthe amino acid sequences of the TK enzymes of the various herpesvirusspecies are dissimilar. In addition, biochemical transformationexperiments in which cloned IBRV DNA fragments are transfected into tk⁻mutant mouse (LM(TK⁻)) or rabbit (RAB(BU)) cells do not yield coloniesof tk⁺ cells containing integrated IBRV DNA sequences (see: Kit, S.,Qavi, H., Hazen, M., Trkula, D., and Otsuka H., Virol. 113:452-464(1981); Otsuka, H., Hazen, M., Kit, M., Qavi, H., and Kit, S., Virol.113:196-213 (1981); Kit, S., Kit, M., Qavi, H., Trkula, D., and Otsuka,H., Biochim. Biophys. Acta 741:158-170 (1983)). Likewise, transfectionof hybrid plasmids containing IBRV DNA fragments into RAB(BU) cells doesnot lead to the transient expression of TK activity, although TKactivity is expressed in a control experiment in which the transfectionof a hybrid plasmid containing the HSV-1 tk gene is used (see: Otsuka,H., Qavi, H., and Kit, S., Antiviral Res. 2:301-311 (1982)).

On the other hand, it has been found in the present invention that theDNA fragment encoding the IBRV tk gene can be identified through markertransfer experiments. It is to be emphasized that it has been found inthe present invention that marker transfer experiments for theidentification of the IBRV tk gene can only be performed if a tk⁻ IBRVmutant, with a low reversion frequency to tk⁺, and a tk⁻ mutant hostcell permissive for IBRV are employed. As discussed in more detailbelow, it has been found in the present invention that the IBRV mutant(IBRV(B8 D53)) described in U.S. patent application Ser. No. 516,179,filed July 21, 1983, and the RAB(BU) cell line, described in Kit, S. andQavi, H., Virol. 130:381-389 (1983), fill these needs. That is,IBRV(B8-D53) is an excellent starting material since it does not revertto tk⁺ even when the virus is propagated in tissue culture in a mediumthat selects for revertants or after two in vivo serial passages incalves. Similarly, RAB(BU) cells are excellent host cells for IBRVreplication and they with a very low frequency, i.e., less revert to tk⁺than 1×10⁻⁷.

Marker transfer experiments, and the in vitro transcription/translationstudies described in the present invention, also permit the delineationof the approximate boundaries of the IBRV tk gene. Thus, for the firsttime, in the present invention, the IBRV DNA subfragment containing thetk gene to be sequenced and, also, the appropriate nucleotide sequencethat could be deleted so as to disrupt the coding sequence of the IBRVtk gene has been identified.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an IBR vaccineeffective in controlling the spread of IBR disease in its variousmanifestations.

Another object of the present invention is to provide an IBR vaccine,wherein the vaccine can be safely and efficaciously administeredintramuscularly, intranasally, or intravaginally.

Still another object of the present invention is to provide an IBRvaccine, wherein the vaccine can be administered safely to calves and topregnant cows in all stages of pregnancy.

A further object of the present invention is to provide an IBR vaccine,wherein the vaccine virus fails to produce any functional TK enzymeactivity as a result of either (i) a deletion in the coding sequence ofthe tk gene, alone or in combination with (ii) an insertion of anoligonucleotide linker sequence in place of the deletion. A stillfurther object of the present invention is to provide an IBRV vaccine,wherein the vaccine virus can replicate efficiently at temperaturesranging from about 30° C. to 40° C., i.e. inclusive oftemperatureresistant mutants.

Another object of the present invention is to provide an IBR vaccine,wherein the vaccine virus cannot revert to tk⁺ and is easily isolatedfrom tk⁺ IBRV.

Still another object of the present invention is to provide an IBRvaccine, wherein the vaccine virus is distinguishable from any fieldstrain virus and from any other BHV-1 vaccine virus.

A further object of the present invention is to provide an IBR vaccine,wherein the animal vaccinated with such does not acquire a dormantinfection with pathogenic field strains.

A still further object of the present invention is to provide a methodfor the production and use of an IBR vaccine, wherein the vaccine viruscannot revert to tk⁺.

In an embodiment of the present invention, these above-described objectshave been met by a highly attenuated IBRV which fails to produce anyfunctional TK as a result of a deletion in the tk gene, and a MLVvaccine for IBR comprising (1) a pharmaceutically effective amount ofsaid virus, and (2) a pharmaceutically acceptable carrier or diluent.

In another embodiment of the present invention, the above-describedobjects have been met by a process for producing a highly attenuatedIBRV which fails to produce any functional TK as a result of a deletionin the tk gene comprising:

(1) Constructing a hybrid plasmid comprising a cloning vector and a DNAfragment of IBRV containing substantially all of the IBRV tk gene;

(2) Co-transfecting, in tk⁺ host cells, the hybrid plasmid of step (1)with infectious DNAffrom a tk IBRV mutagen-induced mutant;

(3) Selecting, in tk host cells, for tk IBRV from the virus produced instep (2);

(4) Deleting DNA sequences from the hybrid plasmid of step (1) such thatless than substantially all of the IBRV tk gene is present;

(5) Co-transfecting, in tk⁺ host cells, IBRV tk⁺ DNA derived from thetk⁺ IBRV obtained in step (3) with the resulting tk⁻ hybrid plasmid ofstep (4); and

(6) Selecting, in tk⁻ host cells, for tk⁻ IBRV from the virus producedin step (5) so as to produce tk⁻ IBRV mutants which fail to produce anyfunctional TK as a result of a deletion in the tk gene.

In still another embodiment of the present invention, an oligonucleotidelinker is inserted in place of the deleted IBRV DNA in step (4) whileretaining IBRV DNA sequences adjacent to each side of the deleted IBRVDNA fragments.

In a further embodiment of the present invention, the tk⁻IBRV-mutagen-induced mutant in step (2) is a temperature-resistantmutant such that the resulting mutant in step (6) is both temperatureresistant and a tk⁻ deletion mutant.

In an additional embodiment of the present invention, theabove-described objects have been met by a process for producing ahighly attenuated IBRV which fails to produce any functional TK as aresult of a deletion in the tk gene comprising:

(1) Constructing a hybrid plasmid comprising a cloning vector and a DNAfragment of IBRV containing substantially all of the IBRV tk gene;

(2) Co-transfecting, in tk⁺ host cells, the hybrid plasmid of step (1)with infectious DNA from a tk⁻ IBRV mutagen-induced mutant;

(3) Selecting, in tk host cells, for tk⁺ IBRV from the virus produced instep (2);

(4) Deleting DNA sequences from the hybrid plasmid of step (1) such thatless than substantially all of the IBRV tk gene is present;

(5) Co-transfecting, in tk⁺ host cells, IBRV tk⁺ DNA derived from thetk⁺ IBRV obtained in step (3) with the resulting tk⁻ hybrid plasmid ofstep (4);

(6) Selecting, in tk⁻ host cell for tk⁻ IBRV from the virus produced instep (5) so as to produce tk⁻ s, tk⁻ IBRV mutants which fail to produceany functional TK as a result of a deletion in the tk gene; and

(7) Propagating the resulting IBRV which fails to produce any functionalTK as a result of a deletion in the tk gene of step (6) at anon-permissive temperature for a temperature-sensitive virus so as toselect for and produce a temperature-resistant IBRV which fails toproduce any functional TK as a result of a deletion in the tk gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates IBRV(BHV-1) Cooper strain HindIII and BamHI DNArestriction maps. These maps are very similar to those of other strainsof IBRV including the Los Angeles strain. The inverted repeat andterminal repeat regions of the DNA are shown as hatched boxes. Theseregions are present in the short (S) segment of the IBRV genome andbracket the short unique sequences of the DNA. The long unique (L)segment of IBRV DNA is also shown. The IBRV tk gene is located at about0.47 map units on the IBRV genome i.e., within the HindIII-A and BamHI-Jfragments. DNA fragments are lettered according to size, with kb aboveeach line and fractionated map distances below each line.

FIG. 2 schematically illustrates, by example, the restrictionendonuclease maps of hybrid plasmids containing a functional IBRV tkgene. Plasmid pLAH-A was derived by inserting the 21.4 Kbp HindIII-Afragment of the Los Angeles strain of IBRV (see FIG. 1 for HindIIIrestriction map) into the HindIII restriction site of bacterial plasmid,pBR322 which is 4.4 Kbp in size. Plasmid pBR322 is tetracyclineresistant(tet^(R)) and ampicillin-resistant (amp^(R)). The insertion inactivatesthe tet^(R) gene, so that amp^(R), tetracycline-sensitive plasmids, likepLAH-A, can be isolated. Hybrid plasmid pMAR-Kpn is a derivative ofplasmid pMAR420 and contains a unique KpnI cloning site (see: Otsuka,H., Hazen, M., Kit, M., Qavi, H., Kit, S., Virol. 113:196-213 (1981)).The black bar and solid line represent, respectively, pBR322 andHerpesvirus tamarinus nucleotide sequences. Hybrid plasmid pLAK wasobtained by transferring the 6.7 Kbp KpnI fragment of IBRV DNA frompLAH-A to the unique KpnI site of pMAR-Kpn, thereby shortening thecloned IBRV DNA sequence by 14.7 Kbp.

FIG. 3 schematically illustrates, by example, the derivation ofadditional tk⁺ and tk⁻ plasmids employed in the present invention.Hybrid plasmid pLATK was derived by StuI and ClaI restriction nucleasecleavage of pLAK and ligation of the excised 5.1 Kbp StuI/ClaI fragmentto the ClaI/PvuII (2.3 kb) fragment of pBR322, which contains theamp^(R) gene. Plasmid pLATK dl NdeI was derived from pLATK by deletionof the 1.2 kb NdeI sequence of pLATK (4.0 to 5.2 map units). PlasmidpLATK dl NdeI dl BglII/NG/SstI wa derived from pLATK dl NdeI by: (i)deleting the 400 bp BglII/SstI sequence (2.3 to 2.7 map units), and thenii) ligating a linker (a 40 bp oligonucleotide which contains5'-GATCT-3' (BglII) and 5'-GAGCT-3' (SstI) cohesive termini) to thelarge fragment of BglII/SstI-cleaved pLATK dl NdeI, so as to produce aclosed-circular hybrid plasmid of 5.8 Kbp. Plasmid pLATK dl NdeI dlBglII/NG/SstI was used in marker transfer experiments with tk⁺ IBRV DNAto obtain an example of the present invention, i.e. IBRV(NG) dl TK clone1.

FIG. 4 illustrates the nucleotide sequence designated LATK16 (2814 bases, of an IBRV DNA fragment which contains the coding region of the IBRVtk gene and flanking sequences thereof. This sequence is the complementof the DNA strand transcribed to produce IBRV TK messenger RNA. TheBglII and SacI(SstI) restriction sites bracket the nucleotide sequencesdeleted from IBRV(NG) dl TK clone 1. The predicted amino acid sequenceof the IBRV TK polypeptide is shown in the 3-letter amino acid codedesignation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention goes beyond a method of merely isolatingattenuated tk IBRV mutants which can be used safely to prevent IBRdisease, but relates to IBRV deletion mutants which fail to produce anyfunctional TK enzyme activity and processes for the production and usethereof. Since the mutants lack part of the DNA sequence coding for TK,reversion to tk does not occur.

In one embodiment, the present invention comprises a highly attenuatedIBRV which fails to produce any functional TK as a result of a deletionin the tk gene, and a MLV vaccine for IBR comprising (1) apharmaceutically effective amount of said virus, and (2) apharmaceutically acceptable carrier or diluent.

Further embodiment of the present invention comprises a process forproducing a highly attenuated IBRV which fails to produce any functionalTK as a result of a deletion in the tk gene comprising:

(1) Constructing a hybrid plasmid comprising a cloning vector and a DNAfragment of IBRV containing substantially all of the IBRV tk gene;

(2) Co-transfecting, in tk⁺ host cells, the hybrid plasmid of step (1)with infectious DNA from a tk⁻ IBRV mutagen-induced mutant;

(3) Selecting, in tk⁻ host cells, for tk⁺ IBRV from the virus producedin step (2);

(4) Deleting DNA sequences from the hybrid plasmid of step (1) such thatless than substantially all of the IBRV tk gene is present;

(5) Co-transfecting, in tk⁺ host cells, IBRV tk⁺ DNA derived from thetk⁺ BRV obtained in step (3) with the resulting tk⁻ hybrid plasmid ofstep (4); and

(6) Selecting, in tk⁻ host cells, for tk⁻ IBRV from the virus producedin step (5) so as to produce tk⁻ IBRV mutants which fail to produce anyfunctional TK as a result of a deletion in the tk gene.

In still another embodiment, the present invention comprises insertionof an oligonucleotide linker in place of the deleted IBRV DNA in step(4) while retaining IBRV DNA sequences adjacent to each side of thedeleted IBRV DNA fragments.

In a further embodiment of the present invention, the tk IBRVmutagen-induced mutant in step (2) is a temperature-resistant mutantsuch that the resulting mutant in step (6) is both temperature resistantand a tk⁻ deletion mutant.

In an additional embodiment of the present invention, theabove-described objects have been met by a process for producing ahighly attenuated IBRV which fails to produce any functional TK as aresult of a deletion in the tk gene comprising:

(1) Constructing a hybrid plasmid comprising a cloning vector and a DNAfragment of IBRV containing substantially all of the IBRV tk gene;

(2) Co-transfecting, in tk⁺ host cells, the hybrid plasmid of step (1)with infectious DNA from a IBRV mutagen-induced mutant;

(3) Selecting, in tk⁻ host cells, for tk IBRV from the virus produced instep (2);

(4) Deleting DNA sequences from the hybrid plasmid of step (1) such thatless than substantially all of the IBRV tk gene is present;

(5) Co-transfecting, in tk⁺ host cells, IBRV tk⁺ DNA derived from thetk⁺ IBRV obtained in step (3) with the resulting tk hybrid plasmid ofstep (4);

(6) Selecting, in host cells, IBRV tk⁻ for tk⁻ IBRV from the virusproduced in step (5) so as to produce tk⁻ IBRV mutants which fail toproduce any functional TK as a result of a deletion in the tk gene; and

(7) Propagating the resulting IBRV which fails to produce any functionalTK as a result of a deletion in the tk gene of step (6) at anon-permissive temperature for a temperature-sensitive virus so as toselect for and produce a temperature-resistant IBRV which fails toproduce any functional TK as a result of a deletion in the tk gene.

The tk gene is approximately 1500 bp in size. The deletion mutants canbe produced by eliminating a 75 to 1500 bp DNA fragment from anappropriate coding region of the tk gene so that proper folding orsubstrate binding of the TK is prevented. Alternatively, the deletionmutants can be produced by eliminating a 10 to 100 bp DNA fragment sothat the proper reading frame of the gene is shifted. In the latterinstance, a truncated polypeptide may be produced because polypeptidesynthesis is aborted due to a frame shift-induced stop codon. Thepreferred size of the deletion is about 75 to 750 bp.

As used herein, "flanking sequences" means the sequences upstream,downstream, or both upstream and downstream, from the tk gene codingsequences. The upstream sequences contain the transcriptional controlsignals, i.e., promoters and enhancers, wherein the downstream sequencescontain the transcription termination and polyadenylation signal of thetk gene.

The precise IBRV tk gene sequences which must be present in the hybridplasmid of step (1) will depend on the sequences chosen for the deletionand the restriction nucleases to be employed in the engineering of thedeletion mutant.

The tk⁻ IBRV mutant employed in this embodiment may also contain one ormore mutagen-induced point mutations in the coding region of the tkgene. Therefore, in this instance, the hybrid plasmid to be employed instep (1) must contain tk IBRV gene sequences to replace the specificsequences mutated in the tk⁻ IBRV mutant. Recombination events betweenthe tk IBRV DNA and the hybrid plasmid of step (1) have to occur bothupsteeam and downstream from the mutagen-induced point mutation(s) inthe tk⁻ IBRV gene. Although the crossover events, i.e., marker rescue,by which the hybrid plasmid of step (1) replaces the mutated tk⁻ BRV DNAmight theoretically occur even when the rescuing plasmid IBRV DNAfragment is small, e.g. 50 to 100 bp; in practice, marker rescue by sucha small DNA fragment is unlikely. By contrast, the probability of markerrescue is greatly increased when the rescuing DNA fragment is greaterthan 1.0 kb. Note, the IBRV DNA insert in pLAH-A, described below, is21.4 kb.

The specific IBRV DNA sequences adjacent to the deletion in the plasmidrequired in step (4) depend on the specifics of the deletion in thehybrid plasmid. In general, the size of the IBRV DNA sequences adjacentto both the 3' and 5' sides of the deletion will be at least about 400bp. In pLATK dl NdeI dl BglII/NG/SstI, described in detail below, the 3'and 5' sequences on both sides of the deletion were 2.3 kb and 1.4 kb inlength.

In a second embodiment, the deletion mutants can contain aoligonucleotide linker in place of the deleted IBRV DNA. Theoligonucleotide linker is generally 8-10 nucleotides in length, but canbe longer, e.g. about 50 nucleotides, or shorter, e.g. 4, 5 or 7nucleotides. The preferred length of the oligonucleotide linker is about20 to 40 nucleotides in length. The DNA sequence of the oligonucleotidelinker is not critical.

The method of inserting the oligonucleotide into the deletion in theplasmid DNA will depend upon the type of oligonucleotide linker used.Palindromic double stranded linkers containing one or more restrictionnuclease sites in the oligonucleotide sequence (New England Biolabs) maybe inserted by well known procedures (see: Maniatis, T., Fritsch, E.F.,Sambrook, J., Molecular Cloning, Cold Spring Harbor Laboratory (1982)).Oligonucleotide linkers may also be inserted into deletions in plasmidDNA by tailing ends with complementary homopolymers using terminaltransferase (see: Maniatis, T., Fritsch, E.F., Sambrook, J., MolecularCloning, Cold Spring Harbor Laboratory (1982)). Alternatively, as in theexample of the present invention, an oligonucleotide linker may beinserted into a deletion in a plasmid by bridging, through annealing ofoligonucleotide containing ends complementary to a cleaved plasmid's 3'essed and 3'-protruding cohesive ends, followed by filling in of the gapcomplementary to the oligonucleotide sequence with DNA polymerase(Klenow's fragment). After subsequent ligation with T4 DNA ligase,closed circular DNA molecules can be regenerated. By the judiciouschoice of oligonucleotide linker length, frame shift mutations may beproduced in the tk gene, augmenting the effect of deletions within thetk gene.

The particular cloning vector employed in the present invention toconstruct a hybrid plasmid comprising a DNA fragment of IBRV containingthe IBRV tk gene and flanking sequences thereof is not critical as longas the cloning vector contains a gene coding for a selective trait, e.g.drug resistance. Examples of such cloning vectors include pBR322 andpBR322-based vectors (see: Sekiguchi, T., Nishimoto, T., Kai, R. andSekiguchi, M., Gene 21:267-272 (1983)), pMB9, pBR325, pKH47 (BethesdaResearch Laboratories), pBR328, pHC79, phage Charon 28 (BethesdaResearch Laboratories, Boehringer Manneheim Biochemicals), pKBll,pKSV-10 (P-L Biochemicals), pMAR420 (see: Otsuka, H., Hazen,

M., Kit, M., Qavi, H. and Kit, S., Virol. 113:196-213 (1981)) and oligo(dG)-tailed pBR322 (Bethesda Research Laboratories).

pBR322 is the preferred cloning vector employed in the present inventionsince the IBRV HindIII-A fragment was found, see below, to contain theIBRV tk gene and pBR322 has only one HindIII cloning site. Insertion ofa DNA fragment at this site inactivates the cloning vector tetracyclinegene, but not the ampicillin gene, so that tetracycline-sensitive,ampicillin-resistant hybrid plasmids that are larger than pBR322 due tothe insertion can easily be isolated.

Other cloning vectors containing unique cloning sites which are usefulin the present invention can be determined upon evaluation ofrestriction nucleases other than HindIII which produce fragmentscontaining the IBRV tk gene. Other restriction nucleases which can beemployed to produce fragments containing the IBRV tk gene, and thusother cloning vectors which can be useful in the present invention, arereadily apparent from the IBRV tk gene sequence and hybrid plasmidsshown in FIGS. 2-4 and which are discussed more fully below.

For example, pMB9, pBR325, pKH47, pBR328, pHC79 and phage Charon 28 alsohave a single HindIII cloning site and can be used to clone theHindIII-A fragment of IBRV. Alternatively, the 3.4 kb PstI fragment ofIBRV (map units 0.2 to 3.6 of pLATK dl NdeI, described below) can becloned at the PstI site of pBR322 or pBR325 with selection ofampicillin-sensitive, tetracycline-resistant transformed bacteria.

Similarly, oligo (dG)-tailed pBR322 can be employed as the cloningvector with an oligo dC)-tailed HindIII fragment of IBRV.

The specific host employed for growing the plasmids of the presentinvention is not critical. Examples of such hosts include E. coli K12RRl (see: Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C.,Heyneker, H.L., Boyer, H.W., Crosa, J.H., and Falkow, S., Gene 2:95-113(1977)); E. coli K12 HB101 (ATCC No. 33694); E. coli MM21 (ATCC No.336780); and E. coli DHl (ATCC No. 33849). E. coli K12 RRl is thepreferred host and has an F hsd R hsd M genotype.

Similarly, alternative vector/cloning systems could be employed such asplasmid vectors which grow in E. coli or Saccharomvces cerevisiae, orboth, or plasmid vectors which grow in B. subtilus (see: Ure, R.,Grossman, L. and Moldave, K., Methods in Enzymology "Recombinant DNA",vol. 101, Part C, Academic Press, N.Y. (1983)).

The specific tk⁺ host cells employed in the present invention are notcritical so long as they allow for permissive growth of IBRV. Examplesof such tk⁺ host cells include RAB-9 (ATCC No. CRL-1414); primary rabbitkidney cells, secondary rabbit kidney cells; rabbit cornea (SIRC) cells(ATCC No. CCL-60), rabbit kidney (LLC-RKl) cells (ATCC No. CCL-106),embryo bovine trachea (EBTR) cells (ATCC No. CCL-44), bovine turbinate(BT) cells (ATCC No. CRL-1390), and bovine kidney (MDBK) cells (ATCC No.CCL-22). (The American Type Culture Collection Catalog indicates thatsome types of lamb, goat, cat, and horse cells may also be permissivefor IBRV(Los Angeles) (ATCC No. VR-188)). RAB-9 are the preferred tk⁺host cells employed in the present invention. However, it should benoted that for the production of virus used for vaccination of animalsin the field, a U.S. Department of Agriculture certified cell linepermissive for IBRV, preferably of the same species as the animal to bevaccinated, and free of other infectious agents, should be used. Forexample, a suitable bovine cell line would be a certified diploidnontumorigenic bovine turbinate or kidney cell line free of mycoplasmaand other viruses.

The specific tk⁻ host cells employed in the present invention are notcritical so long as they allow for permissive growth of IBRV. An exampleof a tk⁻ host cell which allows permissive growth of IBRV is the rabbitRAB(BU) cell line, which was derived from RAB-9 cells (see: Kit, S. andQavi, H., Virol 130:381-389 (1983)). Other tk⁻ host cells of rabbit orbovine origin which can be employed in the present invention can beobtained by following, for example, the procedures previously used toisolate tk⁻ mouse, human, and rabbit cell lines (see: Kit, S., Dubbs,D.R., Piekarski, L.J:, and Hsu, T.C., Exptl. Cell Res. 31:297-312(1963); Kit, S., Dubbs, D.R., and Frearson, P.M. Int. J. Cancer. 1:19-30(1966); and Kit, S. and Qavi, H., Virol. 130:381-389 (1983)). RAB(BU)cells are the preferred tk⁻ host cells employed in the present inventionnot only because they permit the replication to high titers of both tk⁺and tk⁻ IBRV strains, but also because they do not detectably revert totk⁺ in selective medium (hypoxanthine, 10⁻⁴ M; aminopterin, 10⁻⁶ M;thymidine, 4×10⁻⁵ M; and glycine, 10⁻⁵ M (hereinafter "HATG medium"))and they can be used for the plaque titration of IBRV at both permissive(about 34.5° C.) and non-permissive (about 39.1° C.) temperatures. It isimportant that the tk⁻ cells do not detectably revert to tk⁺ in HATGmedium, because reversion to tk⁺ would interfere with autoradiographicand thymidine plaque autoradiographic assays employed to distinguish thephenotypes of tk⁺ and tk⁻ viruses and mixtures thereof.

The specific tk IBRV strain employed in the present invention is notcritical and can be either a non-temperature-resistant or atemperature-resistant strain. Examples of such tk⁻ IBRV strains includethe non-temperature-resistant, 5-bromovinyldeoxyuridine-resistant IBRVmutant of IBRV(P8-2) (see: Weinmaster, G.A., Misra, V., McGuire, R.,Babiuk, J.A., and DeClercq, E., Virol. 118:191-301 (1982)) and thetemperature-resistant IBRV(B8-D53) (see: Kit, S. and Qavi, H., Virol.130:381-389 (1983); and U.S. patent application Ser. No. 516,179, filedJuly 21, 1983; ATCC No. VR-2066). IBRV(B8-D53) is the preferred tk⁻ IBRVstrain employed in the present invention for the following reasons.First, this mutant was obtained by serial passage of the Los Angelesstrain of IBRV in bovine and rabbit cells in the presence of a mutagen,i.e., 5-bromodeoxyuridine, so that multiple genetic alterations haveaccumulated in the IBRV genome. As a result, the ability of the virus tocause disease is reduced. Second, this strain does not detectably revertto tk⁺ in vitro in tissue culture or in vivo in calves. Hence, the viruscan be used in marker transfer studies for the analysis of IBRV DNAfragments containing the coding region of the tk gene. Third, thisstrain replicates in permissive cells and over the temperature range ofabout 30° C. to 40° C., i.e. is temperature resistant. Fourth, pilotexperiments in calves and in pregnant cows using this strain havedemonstrated the safety and efficacy thereof. That is, as describedabove, recent studies have demonstrated that IBRV(B8-D53) is highlyattenuated, can be administered safely by intramuscular, intranasal, orintravaginal injections to calves and to pregnant cows, and protectsthese animals from IBR when the calves and pregnant cows arechallenge-exposed to the highly virulent Cooper strain of BHV-1.

The specific tk⁺ IBRV strain employed in the present invention is notcritical and can be either non-temperature resistant or temperatureresistant. Examples of such tk⁺ IBRV strains include: the followingnon-temperature-resistant strains: Los Angeles strain (ATCC No. VR-188),Cooper strain (ATCC No. VR-864), IPV strain K22 (see: Kendrick, J.W.,Gillespie, J.H., and McEntee, K., Cornell Vet. 48:458-495 (1958)),strains MO3, MO6, BFN-IH, BFN-IIN, BFN-IID, Gi 1 to 5, Bi, B4, BRV, LAE,V3 415, V3 416, V3 18, V3 93 (see: Gregersen, J-P., Pauli, G., andLudwig, H., Arch. Virol. 84:91-103 (1985)), BFA Wabu strain (see:Ackermann, M. and Wyler, R., Vet. Microbiol. 9:53-63 (1984)), strainP8-2 (see: Weinmaster et al., Virol., 118:191-201 (1982)), strains P10,P10, and P34 (see: Engels, M., Steck, F., and Wyler, R., Arch. Virol.67:169-174 (1981)), Alberta (Canada) isolates No. 1 to No. 122 (see:Misra, V., Babiuk, L.A., and Darcel, C. le Q., Arch. Virol. 76:341-354(1983)); or temperature-resistant strains such as IBRV(RTK-lB). Thepreferred tk⁺ IBRV strain employed in the present invention isIBRV(RTK-lB). As described in detail below, this strain was obtaine.d bymarker transfer of the tk gene from a hybrid plasmid to IBRV(B8-D53)(ATCC No. VR-2066), i.e. a tk⁻ IBRV strain which contains multiplemutations. Thus, IBRV(RTK-lB) retains the multiple mutations ofIBRV(B8-D53) and is temperature resistant. IBRV(RTK-lB) differs fromIBRV(B8-D53) only in that IBRV(RTK-lB) expresses functional TK andIBRV(B8-D53) does not.

In the context of this invention, a temperature-resistant virus is avirus which is non-temperature sensitive. Thus, a temperature-resistantvirus is capable of replicating, at a non-permissive temperature, i.e.about 38.5° C. to 40° C., preferably 39.1° C., about as well as theparental virus or field isolates of IBRV replicate at a permissivetemperature. By contrast, temperature-sensitive IBRV strains containmutations in viral genes essential for replication, whereby functionalgene products are produced at permissive temperatures, i.e. about 32° C.to 37.5° C., preferably 34.5° C., but not at non-permissivetemperatures. Therefore, in temperature-sensitive viruses, production ofinfectious virus particles is 4 to 7 logs lower at the non-permissivetemperatures compared to production at permissive temperatures. Withtemperatureresistant virus strains, production of infectious virusparticles is about the same at non-permissive temperatures as atpermissive temperatures.

Some temperature-sensitive respiratory virus strains, for example, atemperature-sensitive mutant of IBRV, have previously been used as MLVvaccines (see: Pastoret, P.P., Thiry, E., Brocphier, B., and Derboven,G., Ann. Rech. Vet. 13:221-235 (1982) and Chanock, R.M., J. Infect. Dis.143:364-374 (1981)). The rationale for such use is that thetemperature-sensitive virus can undergo limited replication at privitgedsites, such as the upper respiratory tract, and elicit local hostimmunological responses. However, the temperature-sensitive viruses areimpaired in replication in the deeper tissues of the host animal, wherethe temperature is non-permissive for virus replication.

Temperature-resistant viruses are superior to temperature-sensitiveviruses as MLV vaccines because: (1) attenuation results fromalterations in specific pathogenic virus genes rather than fromcrippling viral genes required for replication; and (2) thetemperature-resistant virus strains can be administered IM, IN, orintravaginally and can replicate in the deep tissues of the body so asto elicit a more complete and prolonged immunological response.

In contrast, temperature-sensitive viruses only replicate atlow-temperature sites, such as the upper respiratory tract and thus canonly be administered IN.

The possible selection means employed in steps (3) and (6) are notcritical to the present invention and are well known in the art (se U.S.Pat. No. 4,514,497). For example, in step (3) selection can be carriedout using HATG medium and in step (6) selection can be carried out using5-bromodeoxyuridine (hereinafter "BrdUrd"), 5-iododeoxyuridine,5-bromovinyldeoxyuridine or arabinosylthymine.

A pharmaceutically effective amount of the abovedescribed MLV of thepresent invention can be employed along with a pharmaceuticallyacceptable carrier or diluent as a vaccine against IBR in animals, suchas bovine, sheep, goats and swine.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include any physiological buffered medium, i.e,about pH 7.0 to 7.4, containing from about 2.5 to 15% serum which doesnot contain antibodies to IBRV, i.e., is seronegative for IBRV.Agammaglobulin serum is preferred to serum which contains gammaglobulin. Examples of serum to be employed in the present inventioninclude swine serum, fetal calf serum, horse serum and lamb serum.Agammaglobulin calf serum is preferred for the vaccination of calves.Agammaglobulin swine serum from pigs seronegative for IBRV is preferredfor the vaccination of swine. Serum protein such as porcine albumin orbovine serum albumin (hereinafter "BSA") in an amount of from about 0.5to 3.0% can be employed as a substitute for the serum. However, it isdesirable to avoid the use of foreign proteins in the carrier or diluentwhich will induce allergic responses in the animal being vaccinated.

The virus may be diluted in any of the conventional stabilizingsolutions containing phosphate buffer, glutamate, casitone, and sucroseor sorbose, or containing phosphate buffer, lactose, dextran andglutamate.

It is preferred that the vaccine viruses of the present invention bestored at a titer of at least 105 to 10⁶ PFU/ml at -70° C. to -90° C. orin a lyophilized state at 2° C. to 7° C. The lyophilized virus may bereconstituted for use with sterile distilled water or using an aqueousdiluent containing preservatives such as gentamicin and amphotericin Bor penicillin and streptomycin.

The useful dosage to be administered will vary depending upon the age,weight and species of the animal vaccinated and the mode ofadminstration. A suitable dosage can be, for example, about 10⁴.5 to 10⁷PFU/animal, preferably about 10⁴.5 to 10⁵.5 PFU.

The vaccines of the present invention can be administered intranasally,intravaginally or ntramuscularly. Intramuscularly is the preferred modeof administration.

The following examples are provided for illustrative purposes and are inno way intended to limit the scope of the present invention.

In the following examples, all media and buffer solutions were made upin glass distilled water unless otherwise indicated.

EXAMPLE 1 Construction of Hvbrid Plasmids A. Growth Medium for TissueCulture Cells

The tk host cells (RAB-9) cells were propagated in atemperature-controlled, CO₂ incubator, in Eagle's minimum essentialmedium (hereinafter "APMEM") (Flow Laboratories, Inc.) supplemented with10% (v/v) bovine fetal serum (hereinafter "BFS") or 10% (v/v) lambserum, 20 mM bicarbonate, plus 10 mM Hepes (pH 7.3), and 2 mM glutamineplus 50 g/ml neomycin. This medium will be referred to hereinafter as"growth medium". tk⁻ host cells (RAB(BU)) were grown in the same growthmedium as RAB-9 cells, but which was supplemented with BrdUrd (25ug/ml), except as described below for the passage preceding eachexperiment.

B. Purification of IBRV

IBRV DNA was prepared essentially as described by Pignatti et al for thepreparation of HSV DNA (see: Pignatti, P.F., Cassai, E., Meneguzzi, G.,Chemciner, N., and Milanesi, G., Virol. 93:260-264 (1979)).

More specifically, 20 ml -ounce prescription glass bottle monolayercultures of RAB.9 cells (about 5×10⁶ cells/cuture) containing 20 ml ofgrowth medium were infected at a multiplicity of infection (hereinafter"m.o.i.") of 5 PFU/cell of IBRV and incubated for 3 hr at 34.5° C., atwhich time cellular DNA synthesis had been inhibited by the viralinfection. Then 1.0 μCi/m 0.25 ug/ml of (3H)thymidine was added toradioand actively label the viral DNA and incubation was continued at34.5° C. for 17 hr more. The cells were dislodged from the glass byscraping into the growth medium with a rubber policeman, centrifuged at600×g, washed with ice cold phosphate-buffered saline solutioncomprising 0.14 M NaCl, 0.003 M KCl, 0.001 M CaCl₂, 0.0005 M MgCl₂, and0.01 M phosphate, pH 7.5 (hereinafter "PBS"), containing 10 g/mlnon-radioactive thymidine. Next, the cells were centrifuged at 600×g andthen frozen in an ethanol-dry ice bath.

After thawing, the cell pellet (about 0.7 ml) was resuspended in 9volumes of lysing solution comprising 0.25% (w/v) Triton X-100, 10 mMEDTA, 10 mM Tris-HCl, pH 7.9. Next, the cell suspension was transferredto a Dounce homogenizer, and incubated at room temperature for 20-30 minwith gentle mixing.

Then, the cell suspension was transferred to a glass centrifuge tube andNaCl was added to a final concentration of 0.2 M. Next, the tube wasinverted several times, and the solution was immediately centrifuged at1000×g at 4° C. for 10 min.

The resulting supernatant was decanted into a glass tube anddeproteinized by incubating with 100 μg/ml proteinase K (E. M. Science)in buffer solution comprising 10 mM Tris-HCl, pH 7.5, 1.0 mM EDTA(hereinafter "TE buffer") for 1 hr at 37° C. Then, 1 volume of 90% (v/v)redistilled phenol was added, the solution was mixed by inversion,centrifuged at 20,000 x g, and the aqueous phase, i.e., top phase, wastransferred to a polyallomer centrifuge tube. Solid sodium acetate wasthen added to a concentration of 4.0% (w/v), the nucleic acids wereprecipitated with 2 volumes of ice cold ethanol, and incubated overnightat -20° C. Thereafter, the precipitate was collected by centrifugationat 16,000 rpm at 4° C. in a Spinco SW25 rotor, dissolved in 2.0 ml TEbuffer, and dialyzed at 4° C. against TE buffer.

The resulting DNA solution was then transferred to a polyallomercentrifuge tube and CsCl in TE buffer was added to 57% (w/w) (p =1.715g/cm2). Next, the DNA was centrifuged for 46 hr at 22.5° C. at 44,000rpm in a Spinco No. 50 Ti rotor. Then, 12 drop fractions were collectedfrom the bottom of the polyallomer tube and aliquots of 4.0 μl werecounted in a liquid scintillation spectrometer to locate the IBRV DNAcontaining fractions (ρ=about 1.727 g/cm2). When a total of 25 fractionswere collected, generally fractions 13-15 contained the IBRV DNA.

The IBRV DNA-containing fractions were then pooled and dialyzed againstseveral changes of TE buffer at 4° C. for about 24 hr. The concentrationof DNA was determined fluorometrically. The IBRV DNA yield was about 25μg from 10⁸ cells.

The identity of the IBRV DNA was verified by the pattern of restrictionnuclease-digested IBRV DNA fragments obtained after electrophoresis at4° C. in a submarine gel apparatus (Bethesda Research Laboratories,Inc.) as described below.

More specifically, DNA was cleaved with BamHI, SalI, KpnI, or HindIIIrestriction nucleases under the reaction conditions recommended by themanufacturer (New England BioLabs, Inc.). Next, 1/10 volume of asolution comprising 0.4% (w/v) bromphenol blue, 125 mM EDTA, and 50%(v/v) glycerol was added to terminate the reaction, followed by heatingat 65° C. for 10 min. 20 μl aliquots of each sample was applied into thesample wells of the agarose gel and electrophoresis was carried out asdescribed below.

Electrophoresis of restriction nuclease fragments was carried out on0.6% (w/v) agarose slab gels (see: Kit, S., Qavi, H., Dubbs, D.R., andOtsuka, H.. J. Med. Virol. 12:25-36 (1983)) in electrophoresis buffercomprising 30 mM NaH₂ PO₄, 1.0 mM EDTA, 40 mM Tris-Base, pH 8.1(hereinafter "electrophoresis buffer") at 45 volts, 4° C. for about 16hr. After electrophoresis, DNA fragments were stained by soaking the gelin electrophoresis buffer containing 0.5 μg/ml ethidium bromide,visualized over a long wave UV illuminator, and photographed. Therestriction nuclease maps for the HindIII and BamHI fragments ofBHV-1(IBRV) strain (Cooper) are shown in FIG. 1.

IBRV DNA prepared in this manner had an infectivity of about 100 to 1000PFU/μg DNA in the standard transfection assay.

C. Cloning of the IBRV DNA

The HindIII fragments of DNA from IBRV(Los Angeles) were cloned at theHindIII cleavage site of pBR322 by the following procedure.

4.0 μg DNA from IBRV(Los Angeles) was dissolved in cutting buffercomprising 50 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 100μg/ml BSA (hereinafter "HindIII cutting buffer"). The DNA was thendigested at 37° C. for 1 hr with 40 units of HindIII enzyme (New EnglandBioLabs, Inc.). The reaction was terminated by adding an equal volume of90% (v/v) redistilled phenol, mixing, and centrifuging for phaseseparation. After dialysis of the aqueous phase against 1×TE buffer,sodium acetate was added to 0.1 M followed by the addition of 2 volumesof ethanol, and the DNA precipitate was stored at -20° C. overnight. TheDNA precipitate was collected by centrifugation and dissolved in 1×TEbuffer.

The restriction nuclease fragments were then combined in the followingmanner with pBR322 which had been cleaved with HindIII anddephosphorylated:

4.0 μg of HindIII-cleaved IBRV(Los Angeles) DNA was mixed with 0.5 μg ofHindIII digested and dephosphorylated pBR322 DNA (New England BioLabs,Inc.), in 0.05 ml of a solution comprising 50 mM Tris-HCl (pH 7.8), 10mM MgCl₂, 20 mM dithiothreitol, 1.0 mM ATP, and 50 μg/ml BSA(hereinafter called "ligation buffer", and 1000 units of phage T4 DNAligase (New England BioLabs, Inc.), and incubated overnight at 4° C. Thereaction was terminated by adding EDTA to 20 mM and heating at 65° C.for 10 min.

The hybrid plasmid DNA was diluted in TE buffer and used to transform E.coli K12 1 bacteria as described below (see: Bolivar, F., Rodriguez,R.L., Green, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa,J.H., and Falkow, S., Gene 2:95-113 (1977)).

Bacteria were prepared for transformation using CaCl₂ (see: Mandel, M.and Higa, A., J. Mol. Biol. 53:159-162 (1970)). Specifically, anovernight culture at a density of 2.0 (A600) of E. coli K12 RRl was usedto inoculate 200 ml of broth comprising 1.0% (w/v) bactotryptone, 0.5%(w/v) yeast extract, and 0.5% (wvv) NaCl (hereinafter "ML broth"), at abacterial density of 0.02 (A600). The bacteria were incubated for about2 hr until a density of about 0.5 (A600) was achieved. The bacteria werethen pelleted by centrifugation and resuspended in 1/4 volume of cold 50mM CaCl₂. After a 5 min incubation on ice, the bacteria were againpelleted and resuspended in 1/40 the volume of ice cold 50 mM CaCl .

Next, 0.1 ml of the hybrid plasmid DNA, about 10⁻¹⁰⁰ ng, in TE bufferwas added to 0.2 ml of the CaCl₂ -treated bacteria. The mixture was keptat 4° C. for 30 min. Then, the temperature was raised to 37° C. for 5min and 0.3 ml of ML broth was added. Thereafter, incubation wascontinued for 45 min at 37° C. with gentle shaking. Samples were platedon trypticase soy agar plates (BBL Microbiology Systems) supplementedwith 30 μg/ml ampicillin.

Rapid screening of the resulting clones for the desired hybrid plasmidDNA (hereinafter called "rapid screening procedure" was conducted asfollows:

An overnight culture of bacteria containing hybrid plasmid DNA wasinoculated into 5.0 ml of ML broth containing 30 μg/ml ampicillin andincubated at 37√ C. to a density of about 1.5 (A₆₀₀). One ml of thisbacterial culture was then transferred to a 1.5 ml Eppendorfpolypropylene tube and centrifuged in an Eppendorf centrifuge for about1 minute at room temperature to pellet the bacteria. Next, the bacteriawere resuspended in 0.1 ml of lysozyme solution No. 1 comprising 2.0mg/ml egg lysozyme; 50 mM glucose; 10 mM cyclohexanediamine tetraacetate(hereinafter "CDTA"); and 25 mM Tris-HCl buffer, pH 8.0 (hereinafter"lysozyme solution No. 1") and then incubated for 30 min at 4° C. Next,0.2 ml of 0.2 N NaOH plus 1.0% (w/v) sodium dodecylsulfate was added tothe bacterial suspension and the tube was vortexed and kept at 4° C. for5 min. Thereafter, 0.15 ml of 3.0 M sodium acetate, pH 4.8, was added,and the tube was gently inverted, during which time a "clot" of DNAformed. The DNA was kept at 4° C. for 1 hr to allow chromosomal DNA,protein, and high molecular weight RNA to precipitate. Next, theprecipitate was centrifuged in an Eppendorf centrifuge for 5 min at roomtemperature and the clear supernatant fluid, approximately 0.4 ml,containing recombinant plasmid DNA was transferred to a second Eppendorfcentrifuge tube. Then, 21/2 volumes of ethanol (approximately 1.0 ml)were added to the second tube which was placed at -20° C. for 30 min.The precipitated hybrid plasmid DNA was collected by centrifugation for2 min at room temperature in an Eppendorf centrifuge. Then, the hybridplasmid DNA was dissolved in 0.1 ml of 0.1 M sodium acetate, 0.05 MTris-HCl, pH 8.0, reprecipitated with ethano, collected by againcentrifuging, and finally dissolved in 100 μl of 0.1×TE buffer.

Then, a 10 μl aliquot of hybrid plasmid DNA was diluted in 50 μl HindIIIcutting buffer and 2.0 units of HindIII were added. Following adigestion period of 60 min at 37° C., the sample was mixed with 1/10volume of a solution comprising 0.4% (w/v) bromphenol blue, 125 mM EDTA,and 50% (v/v) glycerol, and about 20 μl was applied to a 0.6% (w/v)agarose slab gel for electrophoretic analysis as described above. Thisanalysis revealed whether the hybrid plasmid contained a HindIII insertand, if so, the size, in kb, of the insert (see: Birnboim, H.C. andDoly, J., Nucl. Acids Res. 7:1513-1523 (1973)).

In this manner, a 25.8 Kbp plasmid containing a 21.5 Kbp HindIII insert,which comigrated with the IBRV HindIII-A fragment in agarose gelelectrophoresis, was isolated and designated pLAH-A (see FIG. 2).

For large-scale preparation of hybrid plasmid DNA, 200 times the amountof plasmid-transformed bacteria were processed as compared with thebacteria used to produce hybrid plasmid DNA for the rapid screeningprocedure described above, except that after the first ethanolprecipitation, the sample was treated at 37° C. for 30 min, with 0.5 mgof pancreatic RNase A (Worthington Biochemical Corp.) from a stocksolution comprising 1.0 mg/ml RNase A in 5.0 mM Tris-HCl, pH 8.0, whichhad been heated at 100° C. for 10 min. The treatment was followed by theaddition of 500 μg of proteinase K (E. M. Science) in TE buffer at 37°C. for 30 min. Subsequently, an equal volume of phenol was added, andthe sample was vortexed and centrifuged as described above to separatethe phases. The aqueous phase was then removed, precipitated withethanol, and collected by centrifugation as described above. Theprecipitate was then dissolved in 0.2 ml of TE buffer and layered on a10.4 ml linear 10-40% (w/v) sucrose gradient in 50 mM NaCl, 10 mMTris-HCl, pH 7.5, 1.0 mM EDTA, and was then centrifuged at 4° C. for 20hr at 24,000 rpm in a Spinco SW41 rotor. 15 drop fractions werecollected from the bottom of polyallomer centrifuge tubes into wells ofplastic trays. A total of 35 fractions was obtained. 5 μl aliquots werethen screened by employing agarose gel electrophoresis as describedabove. Fractions containing hybrid plasmid DNA were pooled, dialyzedagainst 0.1×TE buffer, and stored at 4° C. for further studies.

D. Identification by Marker Transfer of the IBRV Fragment Encoding thetk Gene

Homologous recombination in animal cells between purified viral DNAfragments, or by viral DNA fragments amplified by cloning in plasmidvectors, and genomic viral DNA has been used to rescue mutant sequencesin either the DNA fragment or the viral genome. The procedure, known as"marker rescue" or "marker transfer", has been employed to mapspontaneous mutations and mutations induced by mutagens in the entireviral DNA, as well as to "transfer" mutations from hybrid plasmids intogenomic viral DNA (see: Matz, B., Subak-Sharpe, J. H., and Preston, V.G., J. Gen. Virol. 64:2261-2270 (1983)).

Marker transfer procedures were used to identify the IBRV(Los Angeles)HindIII fragment which encodes the tk gene. That is, a mixture ofinfectious IBRV(B8-D53) DNA and candidate hybrid plasmids containingdifferent inserts of IBRV HindIII fragments were co-transfected intoRAB-9 cells by the calcium phosphate precipitate method (see: Graham, F.L. and Van der Eb., A. J., Virol. 52:456-467 (1973)). Specifically, thefollowing sterile solutions were added to a test tube in sequentialorder:

(1) 0.02 ml of a 100 μg/ml solution of IBRV(B8-D53) DNA in TE buffer;

(2) 0.4 ml of a 10 μg/ml solution of recombinant plasmid containingvarious IBRV(Los Angeles) HindIII fragments in 0.1×TE buffer;

(3) 0.25 ml of water;

(4) 0.2 ml of a 100 μg/ml solution of carrier mouse fibroblast (LM(TK⁻))cell DNA in TE buffer;

(5) 0.125 ml of 2.0M CaCl₂ ; and

(6) 1.0 ml of a 2× balanced salt solution comprising 280 mM NaCl, 1.5 mMNa₂ HPO₄, 50 mM Hepes, pH 7.12 (hereinafter "2×BSS")

The resulting solution was mixed by inversion and kept at roomtemperature for 30 min while a DNA-calcium phosphate precipitate formed.Then, 0.5 ml of the suspension containing precipitated DNA-calciumphosphate was added directly to 5.0 ml of growth medium and plated onRAB-9 cells which had been seeded in 60 mm plastic Petri dishes 36 hrearlier. The cells were incubated at 37° C. for 5 hr. Then, fresh growthmedium was added and the cultures were further incubated at 34.5° C. for2-3 days until extensive cytopathic effects occurred. Virus harvestswere made as described above.

The harvests of the above transfections were analyzed by thymidineplaque autoradiography for the presence and proportion of tk⁺ IBRVrescued from IBRV(B8-D53) by pLAH-A (see: Tenser, R. B., Jones, J. C.,Ressel, S. J., and Fralish, F. A., J. Clin. Microbiol. 17:122-127(1983)) as follows:

100 mm plastic tissue culture grade Petri dishes were seeded with1.25×10⁶ RAB(BU) cells in 10 ml of APMEM +10% (v/v) BFS and incubated at37° C. in a humidified CO₂ incubator until the monolayer wassemiconfluent (2 to 3 days). Then the medium was removed by aspiration,and 0.5 ml of thawed and sonicated virus samples in growth medium wasadded at 100 to 1000 PFU/dish and absorbed to the monolayers at 37° C.for 1 hr. The dishes were overlayed with 10 ml of 0.5% (w/v) methylcellulose in growth medium and incubated at 37° C. for 3 days. Themethyl cellulose overlay was removed by aspiration; then the monolayerswere rinsed with a solution comprising 8.0 g NaCl, 0.4 g KCl, 0.1 gglucose and 0.02 g phenol red per liter of water (hereinafter called"GKN") followed by the addition of 5.0 ml of growth medium containing 3μCi of (methyl-¹⁴ C)thymidine (53-59 mCi/mmole) to each dish. At the endof a 6 hr incubation at 37° C., the medium was removed and themonolayers were rinsed with GKN and methanol, and then fixed withmethanol for 1 min at room temperature. The monolayers were subsequentlywashed two times for 5 min each with 5 % (w/v) trichloroacetic acidcontaining 10 μg/ml nonradioactive thymidine, three times for 5 min eachwith 70% (v/v) ethanol, two times for 5 min each with 90% (v/v) ethanol,and two times for 5 min each with 100% ethanol, all at 4° C. Themonolayer was dried, and 5.0 ml of 0.1% (w/v) crystal violet in waterwas added for 5 min followed by rinsing with tap water and drying atroom temperature. The bottoms of the plates were cut out, mounted oncardboard, and placed in a folder with Fuji X-ray film and exposed at-70° C. for 3 days. The film was developed, and the number of dark rimcircles representing isotope incorporation by tk⁺ plaques were countedand compared to the number of total plaques visible on the crystalviolet stained monolayers. As shown in Table 1 below, the tk⁻ mutationof IBRV(B8-D53) was efficiently rescued by pLAH-A that contained theIBRV HindIII-A fragment. In contrast, hybrid plasmids that contained theIBRV HindIII-G and HindIII-B fragments, which map on either side of theHindIII-A fragment (see FIG. 1), did not rescue the tk⁻ mutation ofIBRV(B8-D53). These results demonstrated that the HindIII-G andHindIII-B fragments do not contain sequences covering the mutation siteon the IBRV(B8-D53) tk gene.

                  TABLE 1                                                         ______________________________________                                        Marker Transfer of IBRV tk.sup.+  Gene                                        from Hybrid Plasmid pLAH-A to IBRV(B8-D53)                                                      IBRV titer after                                                  DNA used for                                                                              transfection  tk.sup.+ /tk.sup.-  by                        Group transfection                                                                              (PFU/ml)      autoradiography                               ______________________________________                                        I     IBRV(B8-D53)                                                                              1 × 10.sup.5                                                                           0/300                                              DNA only                                                                      (control)                                                               II    IBRV(B8-D53)                                                                              2 × 10.sup.6                                                                          50/57                                               DNA plus                                                                      plasmid                                                                       pLAH-A DNA                                                              ______________________________________                                    

To enrich for recombinant tk⁺ viruses, the harvests of transfectionswith Platk dl Nda I were passaged in RAB(BU) cells, i.e., tk⁻ cells(see: Kit, S. and Qavi, H., Virol. 130:381-389 (1983)) in growth mediumcontaining HATG (see: Littlefield, J. W., Science 145:709-710 (1964);Littlefield, J. W., Biochim. Biophys. Acta 95:14-22 (1965); andSzybalska, E. H. and Szybalski, W., Proc. Nat. Acad. Sci. USA,48:2026-2034 (1962)) as follows:

The virus harvests of the transfection in RAB-9 cells were sonicated anddiluted 1:500 in growth medium containing HATG, and confluent monolayercultures of RAB(BU) were inoculated with virus at an m.o.i. of about0.01. After a 1 hr absorption at 37° C., fresh growth medium containingHATG was added and the infection was allowed to progress for 48 hr at34.5° C., at which time virus harvests were again made. A secondselection step was conducted in the same manner, except that the viruswas diluted 1:5000. The harvested virus from the second selectionpassage was plaque-purified in RAB-9 cells (see: Kit, S. and Qavi, H.,Virol. 130:381-389 (1983) and Kit, S., Qavi, H., Dubbs, D. R., andOtsuka, H., J. Med. Virol. 12:25-36 (1983)). The resultingplaque-purified viruses were analyzed by thymidine plaqueautoradiography to verify their tk⁻ phenotype and designatedIBRV(RTK-1A) and IBRV(RTK-1B). Then, virus working pools were preparedin RAB-9 cells. The titers of these working pools were about 5×10⁷PFU/ml.

Additional marker transfer experiments were carried out, as describedabove, with infectious IBRV(B8-D53) DNA and plasmids pLATK (describedbelow) and pLATK dl NdeI (described below) (see FIG. 3). Theseexperiments demonstrated that IBRV(B8-D53) was efficiently rescued bothby pLATK and by pLATK dl NdeI. These results indicate that thenucleotide sequences for IBRV tk gene expression were present within the4.1 Kbp IBRV fragment of pLATK dl NdeI.

To confirm that IBRV(RTK-1A) and IBRV(RTK-1B) were indeed IBRV strains,DNA was prepared, as described previously, cleaved with restrictionendonucleases, and analyzed by agarose gel electrophoresis. The HindIIIand BamHI restriction nuclease patterns were similar to those obtainedwith the Los Angeles and Cooper strains of IBRV.

E. Subcloning of pLAH-A: Construction of pLAK

A 6.7 Kbp KpnI restriction fragment from pLAH-A was cloned into the KpnIsite of pMAR-Kpn. pMAR-Kpn (see FIG. 2) is a 6.0 kb plasmid derived frompMAR420 with a single KpnI cleavage site (see: Otsuka, H., Hazen, M.,Kit, M., Qavi, H., and Kit, S., Virol. 113:196-213 (1981)). pMAR-Kpn wasobtained by deleting the 4.3 XhoI to SalI fragment from pMAR420 (seeFIG. 2). Any other plasmid with a single KpnI cleavage site would beequally suitable in the following procedure such as pUC18 and pUC19(Bethesda Research Laboratories) or pKB11 (Pharmacia, Inc.).

Both plasmids were linearized by cleaving 1.0 μg of pLAH-A and 0.1 μg ofpMar-Kpn with 20 units of KpnI in a cutting buffer comprising 6.0 mMNaCl, 6.0 mM Tris-HCl (pH 7.5), 6.0 mM MgCl₂, 1.0 mM dithiothreitol, 100μg/ml BSA (hereinafter "KpnI cutting buffer") during a 1 hr incubationat 37° C. The reaction was stopped by adding CDTA to a finalconcentration of 20 mM and heating at 65° C. for 30 min. Sodium acetatewas added to 0.3M. Then 2 volumes of ethanol was added and the mixturewas stored at -20° C. overnight to allow complete precipitation of DNA.The DNA precipitate was collected by centrifugation. The KpnI cleavedpLAH-A and pMAR-Kpn plasmid DNAs were dissolved in ligation buffer andthen ligated together by T4 DNA ligase, as described previously. E. coliKl strain RRl was then transformed with the resulting plasmids, asdescribed previously, and the plasmid DNA of recombinant clones wasisolated by the rapid screening procedure described above.

The plasmid DNAs of candidate recombinants were treated with KpnI in theKpnI cutting buffer and analyzed by agarose gel electrophoresis asdescribed previously for HindIII. A 12.7 Kbp plasmid with a 6.0 KbppMAR-Kpn fragment and a 6.7 Kbp KpnI fragment derived from pLAH-A wasobtained and designated pLAK. The large-scale preparation of plasmidpLAK DNA was then carried out as described above.

F. Subcloning of pLAK: Construction of pLATK

The 5.1 Kbp StuI to ClaI IBRV DNA fragment of pLAK was cloned into thePvuII to ClaI cleavage sites of pBR322 (see FIGS. 2 and 3). Morespecifically, 1.0 μg of pLAK was added to a reaction buffer comprising100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 6.0 mM2-mercaptoethanol, and 100 μg/ml BSA. The DNA was digested for 1 hr at37° C. with 16 units of StuI. The reaction was terminated by adding CDTAto 20 mM and heating at 65° C. for 30 min. Sodium acetate was added to0.15M and the DNA precipitated with 2 volumes of ethanol. The DNAprecipitate was collected by centrifugation, then redissolved in ClaIcutting buffer comprising 50 mM NaCl, 6.0 mM Tris-HCl (pH 7.9), 6.0 mMMgCl₂, 100 μg/ml BSA (hereinafter called "ClaI cutting buffer"). TheStuI cleaved pLAK was digested for 1 hr at 37° C. with 8 units of ClaI.The reaction was terminated and the DNA collected by ethanolprecipitation as described above.

0.3 μg of pBR322 was added to a cutting buffer comprising 60 mM NaCl,6.0 mM Tris-HCl (pH 7.5), 6.0 mM MgCl₂, 6.0 mM 2-mercaptoethanol, and100 μg/ml BSA. 5 units of PvuII was then added and the DNA digested for1 hr at 37° C. The reaction was terminated and the DNA ethanolprecipitated and collected as above. The PvuII cleaved pBR322 wasdissolved in ClaI cutting buffer and digested with 4 units of ClaI for 1hr at 37° C. The reaction was terminated and the DNA ethanolprecipitated and collected as above.

The ClaI and StuI cleaved pLAK along with the PvuII and ClaI cleavedpBR322 was combined in ligation buffer and ligated by adding 1000 unitsof T4 DNA ligase at 4° C. overnight. The reaction was terminated andtransformation of E. coli Kl RRl carried out as described previously.

Rapid screening as described above resulted in the identification of aplasmid, designated pLATK which had the 5.0 Kbp StuI to ClaI fragment ofpLAK cloned into the PvuII to ClaI sites of pBR322 (see FIG. 3). Workingpools of plasmid pLATK DNA were then prepared as described above.

G. Subcloning of pLATK: Construction of pLATK dl NdeI

The 1.2 Kbp NdeI fragment of pLATK (4.0 to 5.2 map units, see FIG. 3)was deleted by mixing 0.03 μg of pLATK DNA in a cutting buffercomprising 150 mM NaCl, 10 mM Tris-HCl (pH 7.8), 7.0 mM MgCl₂, 6.0 mM2-mercaptoethanol, 100 μg/ml BSA (hereinafter called "NdeI cuttingbuffer"). The DNA was then digested for 1 hr at 37° C. with 2 units ofNdeI. The reaction was terminated by adding CDTA to 20 mM and heating at65° C. for 30 min. Sodium acetate was added to 0.1M, and 2 volumes ofethanol was added followed by storage at -20° C. overnight. The DNAprecipitate was collected by centrifugation, then dissolved in ligationbuffer. The NdeI cleaved pLATK was religated with T4 DNA ligase asdescribed previously followed by transformation of E. coli Kl2 strainRRl and rapid screening of plasmid DNAs as described previously. Theplasmid DNAs of candidate deletion recombinant hybrids were treated withNdeI in NdeI cutting buffer and analyzed by agarose gel electrophoresisas described previously. A 6.2 Kbp plasmid containing only one NdeIcleavage site was isolated and was designated as pLATK dl NdeI (see FIG.3).

H. Construction of Plasmid pLATK dl NdeI dl BglII/NG/SstI

The 0.4 Kbp BglII to SstI fragment of pLATK dl NdeI (see FIG. 3) wasexcised from 1.0 μg of the plasmid by first incubating the DNA with 8units of SstI for 1 hr at 37° C. in SstI cutting solution comprising 50mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 50 mM NaCl. Then the saltconcentration was increased to 100 mM NaCl and 8 units of BglII wasadded. Incubation was continued for an additional hour. The reaction wasstopped by adding CDTA to a final concentration of 20 mM and heating themixture at 65° C. for 30 min. The DNA was ethanol precipitated andcollected by centrifugation as described previously.

The BglII to SstI ends of the plasmid were bridged with a 40-meroligonucleotide linker having the following sequence:

    (5'-GATCTATGAATCAGGTCGCAGGCGAGAATGAGTAAGAGCT-3')

(hereinafter "NG linker"). The NG linker was synthesized byphosphoramidite chemistry on an automated DNA synthesizer (Systec, Inc.)according to the manufacturer's instructions. The BglII and SstI cleavedpLATK dl NdeI plasmid DNA was annealed at 65° C. for 45 min to 8.7 μg ofthe NG linker in an 18 μl reaction mixture comprising 6.6 mM Tris-HCL(pH 7.5), 6.6 mM MgCl₂, 1.1 mM dithiothreitol, 55 mM NaCl, followed byslow cooling at 4° C. in a refrigerator. The single strandedoligonucleotide bridged the cohesive BglII and SstI ends of the plasmid.The gapped sequence complementary to the NG linker was filled by adding1.0 μl of a solution of 2 mM each of dATP, dGTP, TTP, dCTP, and 1.0 μl(2 units) of E. coli DNA polymerase, Klenow fragment (Bethesda ResearchLaboratories)) and incubating 1 hr at 22° C., followed by heatinactivation at 70° C. for 5 min. Ligation of the ends was accomplishedby adding 5.0 μl of 10 mM ATP, 25 μl of 100 mM Tris, pH 7.8, 20 mMMgCl₂, 40 mM dithiothreitol, 100 μg/ml BSA and 1000 units of T4 ligase,and then incubating at 4° C. overnight. The ligation reaction wasterminated by adding 3.0 μl of 0.25M EDTA and heating at 65° C. for 30min.

The ligation mixture was diluted in 1×TE and E. coli Kl2 strain RRl wastransformed as described previously. Ampicillin-resistant colonies werepicked and plasmid DNA purified by the rapid screening proceduredescribed previously. The resulting plasmids, lacking the 0.4 Kb IBRVBglII to SstI fragment, were about 0.4 Kb smaller than pLATK dl NdeI,and were screened for the presence of SstI and BglII sites. The SstIsite was preserved; however, the BglII site was unexpectedly lost. Arepresentative plasmid was analyzed to confirm that the NG linker waspresent by hybridizing a ³² P-labeled NG linker probe to plasmid DNA, asdescribed in detail below. Working preparations of the plasmid,designated pLATK dl NdeI dl BglII/NG/SstI (see FIG. 3), were thenprepared, as described previously.

I. Exonuclease Treatment of a pLATK dl NdeI Derivative to Remove Some ofthe Nucleotide Sequences 5' to the Coding Region of the tk Gene

To delineate the approximate 5' boundaries of a functional IBRV tk gene,a series of plasmids were constructed with nucleotide deletionsextending from the ClaI site of pLATK dl NdeI downstream to a pointbetween the PvuII and BamHI cleavage sites (see FIG. 3, 0 to 1.6 mapunits). The exonuclease III digestion procedure of S. Henikoff (see:Henikoff, S., Gene, 28:351-359 (1984)) was followed.

A linker containing a KpnI cleavage site and EcoRI and ClaI cohesiveends was synthesized with the automated DNA synthesizer described above.The linker was obtained by first making a 15-mer nucleotide sequence(5'-AATTCGGTACCTCAT-3') and a 13-mer nucleotide sequence(5'-CGATGAGGTACCG-3'). These oligonucleotides contain 11 complementarybase pairs. The complementary oligonucleotides were annealed to give adoublestranded DNA fragment with a 5'-EcoRI cohesive end of 4nucleotides, a 3'-ClaI cohesive end of 2 nucleotides, and a KpnIcleavage site in the double-stranded region.

To insert this linker between the EcoRI and ClaI sites of pLATK dl NdeI,the plasmid was digested with ClaI, and then with EcoRI, and thelinearized plasmid was then ligated to the EcoRI-KpnI-ClaI linker byincubating with phage T4 DNA ligase overnight at 4° C. The modifiedpLATK dl NdeI plasmid was designated pLATK dl NdeI(Eco-Kpn-Cla).

The insertion of a unique KpnI site in pLATK dl NdeI(Eco-Kpn-Cla) wascritical for the subsequent exonuclease III deletion procedure. ##STR1##digestion produces a 3' overhanging single-stranded end, which isresistant to exonuclease III digestion. However, after ClaI cleavage,the newly created ClaI end is susceptible to processive exonuclease IIIdigestion. Thus, after KpnI and ClaI digestion, exonuclease IIItreatment of pLATK dl NdeI(Eco-Kpn-Cla) digests sequences clockwise, butthe sequences counterclockwise on the plasmid are not digested.

The exonuclease III digestion of plasmid pLATK dl Nde(Eco-Kpn-Cla) toobtain deletion plasmids was carried out as follows:

5.0 μg of pLATK dl Nde(Eco-Kpn-Cla) in 100 μl of ClaI cutting buffer wasdigested with 20 units of ClaI (New England BioLabs, Inc.) at 37° C. for2 hr. The reaction was stopped by addition of CDTA to 20 mM and sodiumacetate was added to 0.1M followed by heating at 65° C. for 30 min. TheDNA was ethanol precipitated and collected by centrifugation asdescribed above.

The ClaI cleaved plasmid was redissolved in 100 μl of a KpnI cuttingbuffer, then digested by the addition of 20 units of KpnI (New EnglandBioLabs, Inc.) for 2 hr at 37° C. The reaction was terminated, and theDNA precipitated with ethanol and collected as described above.

The ClaI and KpnI cleaved plasmid was redissolved in 66 mM Tris-HCl, pH8.0, 0.6 mM MgCl₂ at a concentration of 100 μg/ml followed by theaddition of 1/10 volume of exonuclease III (Bethesda ResearchLaboratories, Inc.; 65,000 units/ml) and incubated at 37° C. 5.0 μlaliquots were removed at 30 sec intervals and added to 15 μl of asolution comprising 0.2M NaCl, 5.0 mM EDTA, pH 8.0, and mixed. Afterheat inactivation of the exonuclease III activity at 70° C. for 10 min,60 μl of ethanol was added and the DNA was precipitated overnight at-20° C. and collected by centrifugation.

The exonuclease III digested fractions were redissolved in 50 μl of abuffer comprising 0.25M NaCl, 30 mM potassium acetate (pH 4.6), 1.0 mMZnSO₄, 5% (v/v) glycerol, and then digested with 6.8 units of Slnuclease (Boehringer-Mannheim) at room temperature for 30 min. Thereaction was stopped by the addition of 6.0 μl of 0.5M Tris-HCl (pH8.0), 0.125M EDTA. The reaction mixture was extracted with phenol, andthen chloroform, and next, the DNA was precipitated with two volumes ofethanol and collected by centrifugation.

For each of the samples obtained by sequential digestion, the ends weremade flush with Klenow's enzyme. Specifically, the DNA was redissolvedin 1× buffer comprising 6.0 mM Tris, pH 7.5, 6.0 mM MgCl₂, 1.0 mMdithiothreitol, 50 mM NaCl, and digested with 10 units/ml E. coli DNApolymerase, Klenow fragment (Bethesda Research Laboratories, Inc.) for 2min at 37° C. Then, all four deoxynucleoside triphosphates were added to0.1 mM and incubation was continued for an additional 2 min, followed byheating at 70° C. for 5 min, and ethanol precipitation and collection ofthe DNA as described above.

Each of the plasmids was recircularized by selfligation in ligationbuffer containing 1000 units of T4 DNA ligase overnight at 4° C. Thereactions were terminated by adding EDTA to 20 mM and heating at 65° C.for 10 min.

The DNA ligation mixtures were diluted in 1×TE and used to transform E.coli Kl2 RRl as described previously. The resulting transformants werescreened by the rapid screening procedure described above and analyzedby agarose gel electrophoresis as described above. Plasmids which hadretained progressive deletions were selected, and large-scale plasmidDNA preparations made as described previously.

Two of the plasmids, designated pLATK dl NdeI(Exo36) and pLATK dlNdeI(Exo46), were later used for the construction of the phage pSP65derivatives. These derivatives were transcribed and translated in vitroto give 37,000 dalton polypeptides. These studies, to be described indetail below, demonstrated that the translational start signal of theIBRV tk gene and the reticulocyte ribosomal binding sites were justdownstream (3') to the site, labeled "36" of the nucleotide sequenceshown in FIG. 4.

EXAMPLE 2 Nucleotide Sequence of the IBRV tk Gene

Fragments of the IBRV nucleotide sequence of plasmid pLATK dl NdeI (seeFIG. 3) were subcloned in the double-stranded, replicative form (RF) ofphage Ml3mpl8 and Ml3mpl9 (see: Hu, N. T. and Messing J., Gene17:271-272 (1982) and Messing, J. and Vieira, J., Gene 19:269-276(1982)). The use of these two phages permitted the cloning of smalloverlapping DNA fragments in two orientations. Then, replication of therecombinant phage Ml3 derivatives in E. coli Kl2 JM103 bacteria (NewEngland BioLabs, Inc.) resulted in the synthesis of single-strandedphage DNA which could be used for sequencing reactions.

Sequencing reactions were carried out by the conventionaldideoxynucleotide chain termination method (see: Sanger, F., Coulson, A.R., Barrell, B. G., Smith, A. J. H., and Roe, B. A., J. Mol. Biol.143:161-178 (1980); Sanger, F., Nicklen, S., and Goulsen, A. R., Proc.Nat. Acad. Sci. USA 74:5436-5467 (1977)). The reaction mixture containeda single-stranded phage Ml3mpl8 or Ml3mpl9 subclone of IBRV DNA astemplate, either an Ml3 pentadecamer primer (New England BioLabs, Inc.)or synthetic oligonucleotide primer sequences of IBRV DNA made with theautomated DNA synthesizer-Microsyn 1440 (Systec, Inc.) to initiate DNAsynthesis on each of the templates, (α-³² P)-dTTP as the labeledsubstrate, Mg⁺⁺, the appropriate unlabeled deoxyribonucleosidetriphosphates and dideoxyribonucleoside triphosphates, and E. coli DNApolymerase, Klenow fragment (Bethesda Research Laboratories). Afterincubating the reaction mixture for 15 min at 38° C., a chase solutioncontaining non-radioactive deoxyribonucleoside triphosphates was added.The reaction was terminated after 10 min at 38° C. by adding 10 μl of a12.5 mM EDTA solution containing 0.3M sodium acetate and 200 μg of yeasttRNA (Sigma Chemical Co.). The reaction products were precipitated withethanol, dissolved in 10 μl of a solution comprising 90% (v/v)formamide, 30 mM NaOH, 10 mM EDTA, 0.3% (w/v) bromphenol blue, and 0.3%(w/v) xylene cyanol, heated for 1 min at 90° C., and loaded into 8%(w/v) sequencing gels comprising 7.6% (w/v) acrylamide, 0.4% (w/v)bisacrylamide, 0.003% (v/v) TEMED, 0.007% (w/v) ammonium persulfate, and17% (v/v) formamide.

The nucleotide sequence, designated LATKl6, of a 2814 bp XhoI to SalIfragment containing the IBRV tk gene is shown in FIG. 4. Although thenucleotide sequence shown in FIG. 4 was obtained by sequencing the tkgene of IBRV(Los Angeles), due to the high evolutionary conservatism ofthe IBRV tk gene, other tk⁺ IBRV strains, as exemplified above, would beexpected to have tk genes with substantially similar nucleotidesequences, and, thus, as discussed below, the nucleotide sequence inFIG. 4 can be employed to construct additional tk⁻ IBRV deletion mutantsemploying tk⁺ IBRV strains other than IBRV(Los Angeles) as the startingmaterial.

In FIG. 4, the XhoI site (CTCGAG) at the start of the sequencecorresponds to the pLATK dl NdeI XhoI site at 0.5 map units (see FIG.3). The sequence then extends clockwise from the XhoI site to the SalIsite (GTCGAC) of pLATK dl NdeI at 3.4 map units. TA-rich sequences,which might serve as parts of transcriptional control signals, areunderlined at nucleotides 1127 and 1229. A putative "CAAT" box, which,likewise, might be part of a transcriptional control signal, is alsounderlined. A putative translational start signal, i.e., ATG, for theIBRV TK polypeptide is at nucleotide 1292. This ATG codon is the firstATG codon to follow the TTAAAAA sequence at nucleotide 1127, consistentwith the hypothesis (see: Kozak, M. Nucl. Acids Res., 9:5233-5252(1981)) that eukaryotic ribosomes usually initiate protein synthesis atthe AUG closest to the 5' end of an RNA. Kozak has also observed thateukaryotic mRNAs almost always have a purine at position -3 from the ATGor a G at -3, or both. It may be seen that the sequence GCCATGG(nucleotides 1289-1295) conform to this rule. A putative translationalstop signal, i.e., TAA, for the IBRV tk gene is at nucleotide 2366.

The nucleotide sequence of FIG. 4 contains one open reading frame whichcan be translated to a 358 amino acid polypeptide with a molecularweight of 36,903. The predicted amino acid sequence of this polypeptideis shown in the three-letter notation of the IUPAC-IUB Commission onBiochemical Nomenclature (see: Europ. J. Biochem. 5:151-153 (1968)). Thesize of the predicted IBRV TK polypeptide is similar to that of theHSV-1, HSV-2, and Herpesvirus tamarinus TK polypeptides (see: Otsuka H.and Kit, S., Virol. 135:316-330 (1984) and Kit., S., Kit, M., Qavi, H.,Trkula, D., and Otsuka, H., Biochim. Biophys. Acta 741:158-170 (1983)).Furthermore, amino acid residues 10 to 27 of the predicted IBRV TKpolypeptide are homologous to amino acid residues 49 to 66 of the HSV-1and HSV-2 TK polypeptides and amino acid residues 10 to 27 of theHerpesvirus tamarinus TK polypeptide, which appear to represent aconserved ATP-binding pocket of the TK enzyme. Finally, in vitrotranslation products of the transcripts obtained from this sequenceexhibit a molecular weight of about 37,000, consistent with thepredicted size of the IBRV TK polypeptide (see below for details).

The other two reading frames of the IBRV DNA strand shown in FIG. 4 aswell as the three reading frames of the complementary strand containmany translational stop signals and cannot be translated intopolypeptides of 35,000 to 40,000 molecular weight.

Restriction nuclease sites for more than 75 different restrictionnucleases are predicted from the nucleotide sequence shown in FIG. 4.Some of the common restriction nucleases with only one to three cleavagesites in the sequence are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Restriction Nuclease Cleavage Sites Predicted                                 from the Nucleotide Sequence of the IBRV tk Gene                                              Location of first nucleotide                                  Restriction     in sequence                                                   endonuclease    (Nucleotide No.)                                              ______________________________________                                        ApaI            884, 2350                                                     BamHI           1248, 1473, 2438                                              BglII           1759                                                          MluI            490, 2220                                                     PvuI            700, 792, 2408                                                PvuII           928                                                           SacI (SstI)     2102                                                          SalI            170, 2808                                                     SphI            831                                                           XhoI            1, 2183                                                       ______________________________________                                    

It should be noted that unique BglII and SacI (SstI) cleavage sitesoccur at nucleotides 1759 and 2102, respectively. The sequencesbracketed by these cleavage sites were deleted from plasmid pLATK dlNdeI in the isolation of plasmid pLATK dl NdeI dl BglII/NG/SstI (seeFIG. 4). This deletion eliminates about 113 amino acid residues from thecoding sequence of the IBRV tk gene. Furthermore, the ligation of the NGlinker at the BglII/SstI sites of pLATK dl NdeI dl BglII/NG/SstI wouldalso be expected to change the translational reading frame and tointroduce translational stop signals, i.e., TGA and TAA, in all threereading frames, thereby aborting translation of the sequence beyond theBglII cleavage site.

The predictions of restriction nuclease cleavage sites are consistentwith the restriction map of pLATK dl NdeI (see FIG. 3) and provideprecise data on sites that might be used for engineering other deletionmutations in plasmid pLATK dl NdeI, and thus, other tk⁻ deletion mutantsof the present invention.

It should also be noted that deletions can be made in plasmid pLATK dlNdeI by modifications of the procedures described in detail herein. Forexample, it is not essential that the NG linker be ligated to theBglII/SstI termini after endonuclease cleavage of plasmid pLATK dl NdeI.That is, the cleaved plasmid could have been treated with exonucleasesto create "blunt ends", or to widen the deletion gap and then ligatedwith phage T4 DNA ligase in the absence of a nucleotide linker.

EXAMPLE 3 In Vitro Transcription and Translation of the IBRV tk Gene

The 3 Kbp vectors, pSP64 and pSP65, are well known and have beenconstructed for convenient use as standard subcloning vectors and astemplates for highly efficient in vitro transcription (see: Melton, D.A. et al, Nucl. Acids Res. 12:7035-7056 (1984) and Promega Biotech1985/1986 catalogue and applications guide). These vectors contain aphage SP6 promoter cloned into a pUCl2-derived plasmid, immediatelyupstream from a phage Ml3 polylinker, which makes possible a broad rangeof cloning strategies. There are 11 restriction enzyme sites unique tothe polylinker and the polylinker orientation is inverted in the twovectors.

To learn whether the IBRV DNA sequence tentatively identified as the tkgene could be transcribed and translated in vitro to a 37,000 daltonpolypeptide, as predicted (see FIG. 4), IBRV DNA sequences weretransferred from plasmid pLATK dl NdeI(Exo36) and pLATK dl NdeI(Exo46)to plasmid pSP65, as described below. The former plasmid contained thepLATKl6 sequence starting at nucleotide 1090 and the latter starting atnucleotide 1130 (see FIG. 4).

1 μg of pSP65 DNA was mixed with 1.0 μg of pLATK dl NdeI(Exo36) or pLATKdl NdeI(Exo46) and cleaved first with EcoRI in EcoRI cutting bufferdescribed above and then with PstI in PstI cutting buffer comprising 100mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM MgCl₂ and 100 μg/ml BSA, in afinal volume of 20 μl. The reaction was stopped by adding EDTA to afinal concentration of 20 mM and heating at 65° C. for 20 min. Then, 20μl of phenol:chloroform (1 vol:1 vol) was added, the mixture was shaken,and then centrifuged in the Eppendorf centrifuge. The aqueous (upper)phase was transferred to another tube, and 2 volumes of ethanol wereadded to precipitate the cleaved DNAs. The DNA precipitate was washedwith 70% (v/v) ethanol, dried in vacuum, redissolved in 40 μl ofligation buffer containing 400 units of phage T4 DNA ligase (New EnglandBioLabs, Inc.), and incubated overnight at 4° C. The reaction wasterminated by adding 160 μl of TE buffer and heated at 65° C. for 10min.

CaCl₂ -activated E. coli Kl2 RRl cells were next transformed with theligated DNAs and ampicillin-resistant, tetracycline-sensitive colonieswere screened for the presence of a pSP65 derivative containing a 2 KbpEcoRI/PstI insert. The structures of the plasmids, designatedpSP65(Exo36) and pSP65(Exo46) were confirmed by restriction endonucleasemapping with EcoRI, BamHI, BglII, SacI, XhoI, SalI, and PstI.

To prepare transcripts of pSP65(Exo36) and pSP65(Exo46), these plasmidswere linearized by digesting them with SalI and then resuspended at aconcentration of 1.0 μg/ml in distilled water that had previously beentreated with 0.01% (v/v) diethylpyrocarbonate (hereinafter "DEPC") toinactivate any RNase activity. Then, the following procedure was used.

(a) In vitro transcription:

To a sterile DEPC-treated tube was added:

(1) 19 μl water (sterile distilled water, DEPC-treated)

(2) 10 μl 5×transcription buffer (200 mM Tris-HCl, pH 7.5 (measured at37° C.), 30 mM MgCl₂, 10 mM spermidine, 50 mM NaCl)

(3) 5.0 μl of 0.1M dithiothreitol

(4) 2.0 μl RNAsin (40 units/μl) (Promega)

(5) 10 μl of 5×rNTPs (10 mM GTP, 10 mM ATP, 10 mM CTP, 10 mM UTP)(Promega)

(6) 2.0 μl DNA (1.0 μg/μl linear)

(7) 2.0 μl SP6 RNA polymerase (Bethesda Research Laboratories; 5-10units/μg DNA)

The mixture was incubated at 40° C. for 1 hr and 1.0 μl of 100 μg (200units/100 μl DNase I) (Cooper Biomedical; DPRF grade; 11,290 units/5.6ml in 50% (v/v) glycerol; stored at -20° C.) was added, followed by 1.0μl of RNAsin (40 units/μl (Promega)).

The mixture was again incubated at 37° C. for 10 min and then extractedwith an equal volume of phenol:chloroform (1 vol:1 vol) once. 1/10 thevolume of 1.0M potassium acetate, pH 7.0, and 2.2 volumes of ethanolwere added. The suspension was then incubated at -20° C. for at least 2hr.

(b) mRNA capping:

The ethanol precipitated RNA was centrifuged in an Eppendorf centrifugefor 5 min and the supernatant removed by aspiration. The RNA pellet wasdried under vacuum and resuspended in 17.8 μl of sterile DEPC-treatedwater.

The following substances were next added in the order given:

(1) 6.0 μl of 5×capping buffer, comprising: 250 mM Tris-HCl pH 7.9; 6.25mM MgCl₂ ; 30 mM KCl; 12.5 mM dithiothreitol; 500 μg/ml BSA

(2) 1.0 μl RNAsin (40 units/μl (Promega))

(3) 3.0 μl of 1.0 mM S-adenosyl-methionine (Sigma Chemical Co.)

(4) 1.2 μl of 1.0 mM GTP (Promega)

(5) 1.0 μl of guanylyl transferase (Bethesda Research Laboratories)

The reaction mixture, 30 μl, was incubated at 37° C. for 45 min,extracted with phenol:chloroform (1 vol:1 vol), and then the capped RNAtranscript was precipitated by adding 1/10 volume of 1.0M potassiumacetate, pH 7.0, and 2 volumes of ethanol.

(c) Translation of capped mRNA:

Translation of the capped RNA transcript was carried out with thenuclease-treated rabbit reticulocyte lysate (Promega) as follows:

(1) The RNA was sedimented by centrifuging for 5 min in the Eppendorfcentrifuge and the supernatant was removed by aspiration.

(2) The pellet was washed in ethanol and incubated at -20° C. for 30min. The centrifugation step was repeated and the pellet was dried invacuum.

(3) To the RNA pellet was added in a total volume of 50 μl, ice coldsolutions of:

(a) 8.0 μl water (sterile; treated with DEPC)

(b) 1.0 μl RNAsin (40 units/μl; Promega)

(c) 5.0 μl ³⁵ S-methionine (New England Nuclear; 10 μCi/μl)

(d) 1.0 μl of 1.0 mM amino acid mixture (without methionine) (Promega)

(e) 35 μl reticulocyte lysate (Promega)

The reaction mixture was incubated at 30° C. for 1.5 to 2 hrs.

At the end of the incubation period, the translation products weredenatured for polyacrylamide gel electrophoresis.

(d) Denaturing procedure:

Water was added to the reaction volume (50 μl) to give a final volume of200 μl. Then 100 μl of buffer D comprising 0.0625M Tris, pH 6.8, 0.3%(w/v) sodium dodecyl sulfate, 5.0% (v/v) mercaptoethanol, 10% (v/v)glycerol, and 0.001% (w/v) bromphenol blue, was added, the mixture wasboiled for 2 min, and stored at -80° C. until used.

(e) Polyacrylamide gel electrophoresis of protein translation products:

5×electrophoresis buffer consisted of:

(1) 144 g glycine (Calbiochem)

(2) 30 g Trizma (Sigma Chemical Co.)

(3) 5.0 g sodium dodecyl sulfate (Bethesda Research Laboratories)

Polyacrylamide gels were made as follows:

The 3.0% stacking gel consisted of:

(1) 3.17 ml H₂ O

(2) 1.25 ml upper Tris buffer (4×0.5M Tris-HCl, pH 6.8, 0.4% (w/v)sodium dodecyl sulfate)

(3) 0.5 ml acrylamide:bisacrylamide (30:0.8 (w/w))

(4) 75 μl 2.0% (w/v) ammonium persulfate

(5) 5.0 μl TEMED (Sigma Chemical Co.)

The 10% running gel consisted of:

(1) 12 ml H₂ O

(2) 7.5 ml lower Tris (4×1.5M Tris-HCl, pH 8.8, +0.4% (w/v) sodiumdodecyl sulfate)

(3) 10 ml acrylamide:bisacrylamide (30:0.8 (w/w))

(4) 0.6 ml 2.0% (w/v) ammonium persulfate

(5) 15 μl TEMED

(6) 0.5 ml 50% (v/v) glycerol

75 μl aliquots of the translation products were applied to 1.5 mm thickLaemmli gels and electrophoresed at 40 volts, constant voltage, for 16hr at room temperature. The gels were fixed and stained for 30 min atroom temperature with a solution comprising of 50% (v/v) methanol, 10%(v/v) acetic acid, and 0.015% (w/v) coomassie blue, then destained for 2hr at room temperature with a solution comprising of 10% (v/v) aceticacid and 10% (v/v) methanol. The gel was next dried and subjected todirect autoradiography with Fuji X-ray film at -70° C. for 1 day.

When the pSP65(Exo46) and the pSP65(Exo36) plasmids were transcribed andtranslated as described above, a radioactive band with a molecularweight of about 37,000 daltons was detected. Bands were not detectedwhen the RNA transcript was omitted from the translation reaction. Theseresults demonstrate that the nucleotide sequence extending downstreamfrom the point marked "46" (nucleotide 1129 of FIG. 4) to the PstI siteof pLATK dl NdeI (FIG. 3, 3.6 map units) contains an open reading frametranslatable to a polypeptide with a molecular weight of about 37,000.

EXAMPLE 4

Construction of tk⁻ Deletion IBRV Mutant

A. Recombination of Hybrid Plasmid and tk⁻ IBRV DNA

It was shown above that homologous recombination between intact DNA of atk⁻ IBRV strain, i.e., IBRV(B8-D53), and a hybrid plasmid containing thecoding region of the IBRV tk gene, i.e., pLAH-A, resulted in the rescueof a functional tk gene in the recombinant virus, designated IBRV(RTK-lB).

In order to obtain, by homologous recombination, an IBRV deletion mutantin the tk gene, it was necessary to start with the intact DAN of a tk⁺IBRV and a hybrid plasmid containing a deletion in the coding region ofthe tk gene. The progeny virus obtained following this type of crossmainly comprise parental tk⁺ IBRV. Thus, in order to enrich for the tk⁻IBRV recombinants in the harvests, selective media containing BrdUrd wasemployed, since BrdUrd inhibits tk⁺ IBRV replication and favors theoutgrowth of tk⁻ IBRV.

The hybrid plasmid chosen for the construction of a tk⁻ deletion mutantof IBRV was pLATK dl NdeI dl BglII/NG/SstI. However, other hybridplasmids containing larger or smaller flanking sequences adjacent to thecoding sequence of the IBRV tk gene (see FIG. 4) or larger or smallerdeletions in other portions of the tk gene, could be employed to create"deletion" mutations, without departing from the scope and spirit ofthis invention.

The 3.7 Kbp ClaI to NdeI fragment from pLATK dl NdeI dl BglII/NG/SstIwas excised from the hybrid plasmid by adding 10 μg of pLATK dl NdeI dlBglII/NG/SstI to ClaI cutting buffer containing 20 units of ClaI anddigesting at 37° C. for 3 hr, followed by the addition of 100 units ofNdeI in an equal volume of NdeI cutting buffer, augmented with anadditional 100 mM NaCl, and digested at 37° C. for 3 hr. The reactionwas terminated by adding an equal volume of 90% (v/v) phenol, mixed, andcentrifuged for phase separation. After dialyiis of the aqueous phaseagainst 0.1×TE, the DNA was adjusted to 10 μg/ml and filter sterilized.

The tk⁺ IBRV DNA chosen for the recombination step was IBRV(RTK-lB).Since IBRV(RTK-lB) was derived from IBRV(B8-D53), a vaccine strainattenuated through multiple mutations induced by mutagens, theIBRV(RTK-lB) was the preferred virus to other tk⁺ IBRV field strains forthe construction of the deletion mutant. However, as described above,other strains would be equally suitable without departing from the scopeand spirit of this invention.

The construction of the recombinant tk⁻ deletion mutant of IBRV(RTK-lB)was carried out as follows: RAB-9 cells were seeded in 60 mm Petridishes (0.2×10⁶ cells per dish) and incubated at 37° C. for 48 hr. Then,the following sterile solutions were added to a test tube in sequentialorder:

(1) 0.02 ml of a 50 μg/ml solution of IBRV(RTK-1B) DNA in TE buffer;

(2) 0.2 ml of a 10 μg/ml solution of hybrid plasmid pLATK dl NdeI dlBglII/NG/SstI cleaved with ClaI and NdeI;

(3) 0.65 ml of water;

(4) 1.0 ml of 20 μg/ml solution of salmon sperm DNA in 2×Hepes buffersolution comprising 16 g/l NaCl, 0.74 g/l KCl, 0.25 g/l Na₂ HPO₄.2H₂ O,2.0 g/l glucose, 10 g/l Hepes, pH 7.05 (hereinafter "2×Hepes buffersolution");

(5) 0.13 ml of 2.0M CaCl₂

The resulting solution was mixed by inversion and kept at roomtemperature for 30 min while a DNA-calcium phosphate precipitate formed.Then, 0.5 ml of the suspension containing a calcium phosphateprecipitate of DNA was added directly to 5.0 ml of growth medium andplated on RAB-9 cells which had been seeded in 60 mm Petri dishes 48 hrearlier. The cells were incubated at 37° C. for 5 hr. Then the media wasaspirated, and the monolayer rinsed with 5.0 ml of fresh growth media,followed by the addition of 1.0 ml of a solution of 1×Hepes buffersolution plus 15% (v/v) glycerol. After a 3 min incubation at roomtemperature, the solution was aspirated, the monolayer rinsed with mediaagain, and fresh growth media added. The culture were incubated at 34.5°C. for 3 days until extensive cytopathic effects occurred. Virusharvests were made as described above and stored at -80° C. The virusharvest was then titrated in RAB- 9 cells under agar overlay.

The virus harvest from the co-transfection was thawed, sonicated, anddiluted in growth media supplemented with 50 μg/ml BrdUrd. In order toenrich for tk⁻ IBRV deletion mutants, the harvested virus was diluted togive an input multiplicity of 0.1 PFU/cell and passaged in confluentmonolayer cultures of RAB(BU) cells in 8-ounce prescription bottles ingrowth medium supplemented with 50 μg/ml BrdUrd. After a 1 hr absorbtionat 37° C., the infected monolayer cultures were washed three times withGKN. Then, growth medium containing 50 μg/ml BrdUrd was added,incubation was continued at 34.5° C. for 48 hr, and virus harvests weremade.

The harvest of the first selection step was titrated, and a secondselection step carried out as before. The harvest of the secondselection step was titrated in RAB-9 cells, candidate tk⁻ delectionmutants of IBRV were picked at random from plaques, and virus pools wereprepared. In this manner, 96 tk⁻ IBRV deletion mutant candidates wereobtained.

B. Preparation of Probes for Molecular Hybridization

To verify that deletions existed in the tk gene of the tk⁻ IBRV deletionmutant candidates, along with the presence of the NG linker, molecularhybridization experiments with ³² P-labeled probes were carried out:

(1) Preparation of BglII/SacI(SstI) probe:

This probe was made by nick translation of the RF form of phageMl3mpl9(BglII/SacI), which was made by inserting the BglII to SacI(SstI)nucleotide sequence of pLATK dl NdeI into phage Ml3mpl9 (seeYanisch-Perron, C., Vieira, J., and Messing, J., Gene 33:103-119 (1985))as described in detail below. (Also see FIG. 4, LATK16 sequence fromnucleotide 1759-2102).

Ml3mpl9(BglII/SacI) was constructed as follows. First, a mixture of theRF form of phage Ml3mpl9 and pLATK dl NdeI was cleaved with BamHI andSacI. The reaction products were ligated with phage T4 DNA ligase, usedto transform CaCl₂ -activated E. coli JM105 bacteria, and screened fortransformants containing a 629 bp BamHI-BglII-SacI insert (see FIG. 4,nucleotides 1473 to 2102 of LATK16). The Ml3mpl9 recombinant obtained inthis manner was designated phage 19-301.

The RF form of phage 19-301 was prepared and cleaved successively withBamHI and BglII, ligated with phage T4 DNA ligase, and used to transformCaCl₂ -activated E. coli JM105 bacteria. Since BglII and BamHI cleavedDNAs have the same cohesive ends, most of the recombinant phage obtainedfrom this transformation lacked the 286 bp BamHI to BglII fragment ofphage 19-301 and LATK16 sequence from nucleotides 1473 to 1759), butretained the LATK16 sequence from nucleotides 1759 to 2102 (see FIG. 4).The RF phage DNA was prepared from a candidate recombinant phage; thestructure of the phage, designated Ml3mpl9(BglII/SacI), was thenconfirmed by restriction nuclease mapping. ³² P-labeled probes wereprepared by nick translation of the RF form of Ml3mpl9(BglII/Sac), asdescribed below. Alternatively, the RF form of Ml3mpl9(BglII/SacI) wasfirst cleaved with SacI and PstI (PstI is a cloning site on thepolylinker cloning region of Ml3mpl9 upstream from the BglII/BamHIjunction of the IBRV/Ml3mpl9 insert (see New England BioLabs' 1985/1986catalogue, p. 47), and the SacI to PstI fragment was purified bycentrifugation in a 10-40% (w/v) sucrose gradient at 39,000 rpm for 20hr and nick-translated.

To 25 μl of reaction mixture containing 6.0 μmol PBS, pH 7.4; 1.8 nmoldATP; 1.8 nmol dGTP; 0.1 mCi (α-³² P)dTTP (400 Ci/mmole); 0.1 mCi (α-³²P)dCTP (400 Ci/mmole) (Amersham Corporation), about 1.0 μg plasmid DNAwas added. Then, 1.33 ng in 1.0 μl of DNase I (Worthington BiochemicalCorporation) was added and the reaction mixture was allowed to stand for1 min at room temperature. Next, the reaction mixture was incubated at14° C. with 5.0 units in 1.0 μl of E. coli DNA polymerase I(Boehringer-Mannheim Biochemicals). When the specific activity becamehigher than 2×10⁸ cpm/μg DNA, i e., about 3 hr, the reaction wasterminated by adding 10 μl of 0.25M EDTA (pH 7.4) and heating at 68° C.for 10 min. Then, as carrier, 50 μl of a solution comprising 5.0 mg/mlsonicated salmon sperm DNA in TE buffer, was added to the mixture andthe nick-translated DNA was purified by Sephadex G50 (fine) columnchromatography using 10 mM NaCl, 10 mM Tris-HCl, pH 7.5, 2.0 mM EDTA asthe elution buffer.

The resulting ³² P-labeled, nick-translated DNA was used as a probe inDNA-DNA hybridization experiments after boiling in a water bath for 20min, and quickly cooling on ice to form single-stranded DNA (see: Rigby,P. W. J., Dieckmann, M., Rhodes, G., and Berg, P., J. Mol. Biol.113:237-251 (1977)).

(2) pLATK dl NdeI Probe:

This probe was made by nick translation of plasmid pLATK dl NdeI (seeFIG. 3).

(3) NG Probe Linker:

Synthesis of the NG linker has been described above. To prepare a probe,the NG linker was labeled with ³² P as follows:

50 picomoles of NG linker was added to a reaction mixture comprising 150μCi γ-³² P ATP, 70 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 5.0 mMdithiothreitol, and 5 units of T4 polynucleotide kinase (New EnglandBioLabs, Inc.). The mixture was incubated for 1 hr at 37° C. and thereaction was terminated by adding EDTA to 20 mM, followed bypurification of the labeled NG linker by gel filtration on gel P4(Bio-Rad, Inc.) to remove the unreacted γ-³² P ATP. The elution bufferwas the same as that used for Sephadex G-50 chromatography discussedabove. The probe was not heat-treated before use, since it was alreadysingle-stranded.

C. Identification of Recombinant tk⁻ IBRV Deletion Mutant ofIBRV(RTK-1B) By Molecular Hybridization

Viral DNAs prepared from the candidate recombinants described previouslywere analyzed by the dot blot method (see: Brandsma, J. and Miller, G.,Proc. Nat. Acad. Sci. USA 77:6851-6855 (1980)) to identify viruses thatlacked the 0.4 Kbp BglII to SstI IBRV tk gene fragment. Specifically,24-well multiwell tissue culture trays containing confluent monolayersof RAB-9 cells were infected with 0.05 ml of undiluted candidate virusand incubated at 34.5° C. for 8 hr. The virus inoculum was aspirated,the wells rinsed with 1.0 ml of GKN, and 0.2 ml of 0.5 N NaOH was addedto each well to lyse the cells and release the DNA. After storage atroom temperature overnight, 0.3 ml of 1.0M Tris-HCl (pH 7.5) and 0.5 mlof 20×SSC buffer comprising 3.0M NaCl, 0.3M sodium citrate, pH 7.0(hereinafter "20×SSC") were added per well. For dot blot analysis,nitrocellulose filters in a 96-well Schleicher and Schuell filtrationapparatus was used. The filters were washed with water and with 1×SSCprior to the addition of the DNA samples. To bake the DNA samples to thefilters, the nitrocellulose filters were dried, heated overnight at 60°C. in a vacuum desiccator, and then heated for 2 hr at 80° C. The filterwas placed in a plastic sealable pouch containing 50 ml of 3×SSC, 0.02%(w/v) Ficoll, 0.02% (w/v) BSA, 0.02% (w/v) polyvinylpyrrollidone, 50μg/ml of boiled and alkali-denatured salmon sperm DNA (hereafter called"modified Denhardt's solution"), 10 μg/ml poly(A) and incubatedovernight at 60° C. with shaking. Alkaline salmon sperm DNA was addedfrom a stock solution of about 5.0 mg/ml prepared by dissolving 50 mg ofsalmon sperm DNA in 10 ml of 0.2N NaOH, heating at 100° C. for 20 min todenature and shearing the DNA to about 0.4 kb segments, and thenneutralizing with 0.2 ml of 10 N HCl.

The modified Denhardt's solution was then replaced with 50 ml ofhybridization buffer comprising 50% (v/v) formamide, 0.6M NaCl, 0.2MTris-HCl, pH 8.0, 0.02M EDTA, 0.1% (w/v) sodium dodecylsulfate, 50 μg/mlalkali-denatured salmon sperm DNA, and 10 μg/ml poly(A) (hereaftercalled "hybridization buffer"). Next, air bubbles were squeezed out ofthe bag which was then sealed using an Oster Touch-a-Matic Bag Sealerand incubated at 37° C. for 1 hr on a shaker.

Thereafter, about 1.0 ml, containing about 10⁷ cpm and 50 ng, ofsingle-stranded (³² P) nick-translated BglII/SacI probe, obtained asdescribed above, was added to the bag with a 3.0 ml syringe by piercingthe side of the bag at a corner. Next, the bag was resealed andincubated at 37° C. for up to 48 hr on a shaker to allow forhybridization.

After hybridization had been accomplished, the bag was cut and thesolution was decanted. The filter was then carefully removed and placedinto a tray containing about 100 ml of hybridization buffer containing50 μg/ml denatured salmon sperm DNA for the first wash only, but nopoly(A) in any wash. The filter was washed for 30 min at 37° C. fivetimes with gentle shaking. Next, the filter was washed for 30 min at 37°C. with 0.3×SSC and then placed on filter paper to dry overnight at roomtemperature.

For autoradiography, the filter was replaced on a thin piece ofcardboard covered with Saran-Wrap, and exposed to Fuji X-ray film withan intensifying screen for periods of 5 hr to 2 days a -70° C. About 35out of the 96 candidate viruses did not hybridize to the BglII/SacIprobe, indicating that these clones had a deletion in the BglII toSacI(SstI) sequence of the IBRV -tk gene. Two clones were saved forfurther analysis and designated IBRV(NG) dl TK clone 1 and IBRV(NG) dlTK clone 5.

Viral DNA of high purity was prepared as described above. 0.5 μg ofviral DNA from each of the candidate deletion mutants was cleaved withrestriction nucleases, HindIII, BamHI, KpnI, and SalI, under conditionsspecified by New England BioLabs, and the fragments were separated byelectrophoresis on 0.6% (w/v) agarose at 35 volts (constant voltage) for16 hr at 4° C. The electrophoresis buffer was 0.04M Trizma base, pH 8.1,0.03M NaH₂ PO₄, and 0.001M EDTA. Restriction nuclease fragments of theparental IBRV(RTK-1B) and marker fragments obtained by HindIII digestionof phage lambda DNA and HaeIII digestion of phage ΦX174 RF DNA were alsoelectrophoresed. The gels were stained with ethidium bromide andphotographed as described above to reveal the DNA fragments.

The results demonstrated that the 6.7 Kbp KpnI and the 2.8 Kbp SalIfragments of IBRV(RTK-1B) (see FIG. 2, plasmid pLAK map units 1.6 to8.3, and FIG. 3, plasmid pLATK dl NdeI map units 0.6 to 3.4) werespecifically altered, and that new, more rapidly migrating (smaller)fragments were present in the deletion mutant samples. This isconsistent with the deletion of the approximately 0.4 BglII/SacIsequence from the IBRV tk gene. As expected, no mobility shifts wereapparent in the HindIII-A fragments of the deletion mutants because thesmall deletion was obscured by the large size (21.4 Kbp) of theHindIII-A fragment. Likewise, a 1.1 Kbp BamHI fragment (BamHI-Jfragment) of IBRV(RTK-1B) (see FIG. 3, plasmid pLATK dl NdeI map units2.0 to 3.1) was about 0.4 kb smaller in the deletion mutant DNAfragments than in the BamHI fragments of IBRV(RTK-1B).

After electrophoresis, the separated DNA restriction fragments in theagarose gel were transferred to nitrocellulose filters (Schleicher andSchuell) for molecular hybridization experiments in the followingmanner: The agarose gel was placed in a glass baking tray containing1.0M KOH for 30 min at room temperature and, then, in a glass bakingtray containing 1.0M Tris-HCl, pH 7.0 and 0.6M NaCl for 60 min at roomtemperature. The treated gel was then transferred to a blot apparatus(Bethesda Research Laboratories).

A nitrocellulose filter was prewetted in water for 10 min and then in20×SSC for 5 min. Next, the filter was placed on the gel. Using 20×SSCas the transfer fluid, blotting was allowed to proceed for about 24 hr.The adherent gel was removed from the nitrocellulose filter, and thefilter was rinsed with 6×SSC, dried at room temperature for severalhours, and then in a vacuum desiccator overnight at 60° C. This wasfollowed by 2 hr of baking at 80° C. The nitrocellulose filters wereremoved from the desiccator and placed in Dazey Seal-a-Meal cooking bags(see: Southern, E. M., J. Mol. Biol. 98:503-513 (1975)).

The filter was first pretreated overnight at 60° C. with 50 ml ofmodified Denhardt's solution and hybridization buffer at 37° C. asdescribed previously.

The nitrocellulose filters from three separate gels were next hybridizedto three separate ³² P-labeled probes: (i) BglII/SstI probe; (ii) pLATKdl NdeI probe; and (iii) NG linker probe. The procedure for molecularhybridization of the probes to the nitrocellulose filters and thewashing step were the same as described previously, except that in thecase of the ³² P-labeled NG linker probe, the hybridization reaction wascarried out in 35% (v/v) formamide, 0.6M NaCl, 20 mM EDTA, 0.2M Tris, pH8.0, 0.1% (w/w) sodium dodecylsulfate solution and the final wash waswith 6×SSC.

The results demonstrated that the BglII/SstI probe hybridizedspecifically to a 21.4 kb HindIII fragment, a 1.1 kb BamHI fragment, a6.7 kb KpnI fragment and a 2.8 kb SalI fragment of IBRV(RTK-1B) DNA butnot to fragments of either HindIII-, BamHI-, KpnI- or SalI-cleavedIBRV(NG) dl TK clones 1 and 5 DNA.

In addition, the results demonstrated that the pLATK dl NdeI probehybridized specifically to: (1) an approximately 21 kb HindIII fragmentof IBRV(RTK-1B) DNA and to a 21 kb HindIII fragment of IBRV(NG) dl TKclones 1 and 5 DNA; (2) 17.3 kb, 15.7 kb and 1.1 kb BamHI fragments ofIBRV(RTK-1B) DNA and to 17.3 kb, 15.7 kb and 0.7 kb BamHI fragments ofIBRV(NG) dl TK clones 1 and 5 DNA; (3) a 6.7 kb KpnI fragment ofIBRV(RTK-1B) DNA and to a 6.3 kb KpnI fragment of IBRV(NG) dl TK clones1 and 5 DNA; and (4) 2.8 kb and 0.8 kb SalI fragments of IBRV(RTK-1B)DNA and to 2.4 kb and 0.8 kb SalI fragments of IBRV(NG) dl TK clones 1and 5 DNA. The 1.1 kb BamHI, 6.7 kb KpnI and 2.8 kb SalI fragments ofIBRV(RTK-1B) DNA, which contain the tk gene, were larger by 0.4 kb thanthe corresponding fragments of IBRV(NG) dl TK clones 1 and 5 DNA; i.e.,the 0.7 kb BamHI, 6.3 kb KpnI and 2.4 kb SalI fragments. There was noapparent change in the about 21 kb HindIII fragment of IBRV(NG) dl TKclones 1 and 5 DNA because of the large size of the HindIII-A fragmentrelative to the size of the introduced deletion.

Further, the results demonstrated that the NG linker probe hybridizedspecifically to the about 21 kb HindIII, 0.7 kb BamHI, 6.3 kb KpnI and2.4 kb SalI fragments of IBRV(NG) dl TK clones 1 and 5 DNA, but did nothybridize to the fragments of IBRV(RTK-1B) DNA.

These experiments conclusively demonstrate that IBRV(NG) dl TK clones 1and 5 genomes had a BglII/SstI deletion of about 0.4 kb and an NG linkerinsertion in the IBRV tk gene. IBRV(NG) dl TK clone 1 has been depositedwith the American Type Culture Collection under ATCC No. VR-2112.

EXAMPLE 5 TK Activity of IBRV Strains

To verify that deletion mutant IBRV(NG) dl TK clone 1 lacked the abilityto induce functional TK activity, TK induction experiments were carriedout. More specifically, subconfluent monolayer cultures of RAB(BU) cellswere mock-infected or infected for 7 hr with the IBRV(Los Angeles) orthe IBRV(NG) dl TK clone 1 strains. Cytosol fractions were prepared andassayed for TK activity (see: U.S. Pat. No. 4,514,497 and Kit, S. andQavi, H., Virol. 130:381-389 (1983)). The results obtained are shown inTable 3 below.

                  TABLE 3                                                         ______________________________________                                        Cytosol Thymidine Kinase Activity of RAB(BU) Cells                            Infected for 7 Hr with tk.sup.+  IBRV and tk.sup.-  IBRV Strains                               TK activity                                                                   (picomoles dTMP formed                                       IBRV strain used to infect                                                                     in 10 min at 38° C. per                               RAB(BU) cells    μg protein)                                               ______________________________________                                        Mock-infected RAB(BU)                                                                          0.04                                                         IBRV(Los Angeles)                                                                              1.74                                                         IBRV(NG) dl TK clone 1                                                                         0.03                                                         ______________________________________                                    

Table 3 above demonstrates that: (i) RAB(BU) cells, i.e., tk⁻ rabbitskin cells, have negligible TK activity; (ii) TK activity is acquired byRAB(BU) cells after infection by tk⁺ IBRV(Los Angeles), but not afterinfection by the tk⁻ deletion mutant, IBRV(NG) dl TK clone 1. Thus,deletion of the BglII/SacI fragment from the IBRV tk gene results intota loss of TK-inducing activity.

EXAMPLE 6 Temperature Resistance of IBRV Strains

Replicate, subconfluent monolayer cultures of RAB-9 cells were infectedwith IBRV(NG) dl TK clone 1 at an input multiplicity of about 0.01PFU/cell and incubated in a CO₂ -incubator at 30° C., 34.5° C., and39.1° C. Virus harvsts were prepared at 4 hr after infection at 34.5° C.to determine the amount of infectious virus present immediately afterthe absorption and penetration of the input virus, and after 10 days, 4days and 3 days of incubation, respectively, at 30° C., 34.5° C., and39.1° C. Virus harvests were plaque titrated at 34.5° C. and at 39.1° C.in RAB-9 cells as described in U.S. Pat. No. 4,514,497. The results areshown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Replication of IBRV(NG) dl TK clone 1                                                                   Virus Yield                                                                   (PFU/ml)                                                                      when plaque titrated                                Temperature of                                                                           Time Postinfection                                                                           at:                                                 Virus Growth                                                                             Harvested      34.5° C.                                                                        39.1° C.                            ______________________________________                                        34.5° C.                                                                          4        hr        1.8 × 10.sup.2                                                                 1.4 × 10.sup.2                     34.5° C.                                                                          4        days      2.3 × 10.sup.7                                                                 2.1 × 10.sup.7                     30° C.                                                                            10       days      1.1 × 10.sup.6                                                                 1.3 × 10.sup.6                     39.1° C.                                                                          3        days      6.0 × 10.sup.6                                                                 8.0 × 10.sup.6                     ______________________________________                                    

Table 4 demonstrates that after 4 days of incubation at 34.5° C.,IBRV(NG) dl TK clone 1 multiplied from about 10² PFU/ml to about 2×10⁷PFU/ml and that this titer was attained regardless of whether the plaquetitration was performed at 34.5° C. or 39.1° C. This clearly indicatesthat the virus is temperature resistant. Table 4 also demonstrates thattiters of about 10⁶ and 10⁷ PFU/ml, respectively, were obtained whenharvests were made at 10 and 3 days, respectively, at 30° C. or 39.1° C.Again, the same titers were formed regardless of whether the plaquetitration was at 34.5° C. or 39.1° C. Thus, it is clear that this viruscan grow to high titers over a broad range of temperatures,specifically, from 30° C. to 39.1° C. or higher.

While this invention has been described in detail with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

We claim:
 1. A modified live-virus vaccine for infectious bovinerhinotracheitis comprising:(1) a pharmaceutically effective amount of aninfectious bovine rhinotracheitis virus which fails to produce anyfunctional TK as a result of a deletion in the tk gene; and (2) apharmaceutically acceptable carrier or diluent.
 2. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 1, wherein said deletion is 10 to 1500 bp in size. 3.The modified live-virus vaccine for infectious bovine rhinotracheitisvirus as claimed in claim 2, wherein said deletion is 75 to 750 bp insize.
 4. The modified live-virus vaccine for infectious bovinerhinotracheitis virus as claimed in claim 1, wherein said virus istemperature resistant.
 5. The modified live-virus vaccine for infectiousbovine rhinotracheitis virus as claimed in claim 1, wherein anoligonucleotide linker is present in the deletion site of the tk gene.6. The modified live-virus vaccine for infectious bovine rhinotracheitisvirus as claimed in claim 5, wherein said linker is about 7 to 50nucleotides in length.
 7. The modified live-virus vaccine for infectiousbovine rhinotracheitis virus as claimed in claim 1, wherein said virushas the identifying characteristics of IBRV(NG) dl TK clone 1 (ATCC No.VR-2112).
 8. The modified live-virus vaccine for infectious bovinerhinotracheitis virus as claimed in claim 1, wherein saidpharmaceutically acceptable carrier or diluent is physiological bufferedmedium containing about 2.5 to 15% serum which does not containantibodies to IBRV.
 9. The modified live-virus vaccine for infectiousbovine rhinotracheitis virus as claimed in claim 8, wherein said serumis selected from the group consisting of swine serum, fetal calf serum,horse serum and lamb serum.
 10. The modified live-virus vaccine forinfectious bovine rhinotracheitis virus as claimed in claim 1, whereinsaid pharmaceutically effective amount is about 10⁴.5 to 10⁷ PFU. 11.The modified live-virus vaccine for infectious bovine rhinotracheitisvirus as claimed in claim 10, wherein said pharmaceutically effectiveamount is about 10⁴.5 to 10⁵.5 PFU.
 12. A modified live-virus vaccinefor infectious bovine rhinotracheitis comprising:(1) a pharmaceuticallyeffective amount of an infectious bovine rhinotracheitis virus whichfails to produce any TK as a result of a deletion in the tk gene,produced by a process comprising:(a) constructing a hybrid plasmidcomprising a cloning vector and a DNA fragment of IBRV containingsubstantially all of the IBRV tk gene; (b) co-transfecting, in tk⁺ hostcells, the hybrid plasmid of step (a) with infectious DNA from a tk⁻IBRV mutagen-induced mutant; (c) selecting, in tk⁻ host cells, for tk⁺IBRV from the virus produced in step (b); (d) deleting DNA sequencesfrom the hybrid plasmid of step (a) such that less than substantiallyall of the IBRV tk gene is present; (e) co-transfecting, in tk⁺ hostcells, IBRV tk⁺ DNA derived from the tk⁺ IBRV obtained in step (c) withthe resulting tk⁻ hybrid plasmid of step (d); and (f) selecting, in tk⁻host cells, for tk⁻ IBRV from the virus produced in step (e) so as toproduce tk⁻ IBRV mutants which fail to produce any functional TK as aresult of a deletion in the tk gene, and (2) a pharmaceuticallyacceptable carrier or diluent.
 13. The modified live-virus vaccine forinfectious bovine rhinotracheitis virus as claimed in claim 12, whereinsaid deletion is about 10 to 1500 bp in size.
 14. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 13, wherein 75 to 750 bp in size.
 15. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 12, wherein the tk⁻ IBRV mutagen-induced mutant in step(b) is a temperature-resistant mutant such that the resulting mutant instep (f) is both temperature resistant and a tk⁻ deletion mutant. 16.The modified live-virus vaccine for infectious bovine rhinotracheitisvirus as claimed in claim 12, additionally comprising step (g):(g)propagating the resulting IBRV which fails to produce any functional TKas a result of a deletion in the tk gene of step (f) at a non-permissivetemperature for a temperature-sensitive virus so as to select for andproduce a temperature-resistant IBRV which fails to produce anyfunctional TK as a result of a deletion in the tk gene.
 17. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 12, wherein an oligonucleotide linker is present in thedeletion site of the IBRV DNA of step (d) while retaining IBRV DNAsequences adjacent to each side of the deletion site.
 18. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 17, wherein said linker is about 7 to 40 nucleotides inlength.
 19. The modified live-virus vaccine for infectious bovinerhinotracheitis virus as claimed in claim 12, wherein said cloningvector is selected from the group consisting of pBR322, pMB9, pBR325,pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10, pMAR420 and oligo(dG)-tailed pBR322.
 20. The modified live-virus vaccine for infectiousbovine rhinotracheitis virus as claimed in claim 19, wherein saidcloning vector is pBR322.
 21. The modified live-virus vaccine forinfectious bovine rhinotracheitis irus as claimed in claim 12, whereinsaid hybrid plasmid of step (a) is pLATK dl NdeI.
 22. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 12, wherein in step (d) the IBRV DNA sequences adjacentto each side of the deletion are at least 400 bp in size.
 23. Themodified live-virus vaccine for infectious bovine rhinotracheitis virusas claimed in claim 12, wherein the resulting hybrid plasmid of step (d)is pLATK dl NdeI dl BgII/NG/SstI.
 24. The modified live-virus vaccinefor infectious bovine rhinotracheitis virus as claimed in claim 12,wherein said tk⁺ host cells are selected from the group consisting ofRAB-9 (ATCC No. CRL-1414); primary rabbit kidney cells, secondary rabbitkidney cells; rabbit cornea (SIRC) cells (ATCC No. CCL-60), rabbitkidney (LLC-RKl) cells (ATCC No. CCL-106), embryo bovine trachea (EBTR)cells (ATCC No. CCL-44), bovine turbinate (BT) cells (ATCC No.CRL-1390), and bovine kidney (MDBK) cells (ATCC No. CCL-22).
 25. Themodified live-virus vaccine for infectious bovine rhinotracheitis virusas claimed in claim 24, wherein said tk⁺ host cells are RAB-9.
 26. Themodified live-virus vaccine for infectious bovine rhinotracheitis virusas claimed in claim 12, wherein the tk⁻ IBRV mutagen-induced mutant isselected from the group consisting of the non-temperature-resistantbromovinyldeoxyuridine-resistant IBRV mutant of IBRV(P8-2) and thetemperature-resistant IBRV(B8-D53) (ATCC No. VR-2066).
 27. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 26, wherein the tk⁻ IBRV mutagen-induced mutant is thetemperature-resistant IBRV(B8-D53) (ATCC No. VR-2066).
 28. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 12, wherein said pharmaceutically acceptable carrier ordiluent is physiological buffered medium containing about 2.5 to 15%serum which does not contain antibodies to IBRV.
 29. The modifiedlive-virus vaccine for infectious bovine rhinotracheitis virus asclaimed in claim 28, wherein said serum is selected from the groupconsisting of swine serum, fetal calf serum, horse serum and lamb serum.30. The modified live-virus vaccine for infectious bovinerhinotracheitis virus as claimed in claim 12, wherein saidpharmaceutically effective amount is about 10⁴.5 to 10⁷ PFU.
 31. Themodified-live virus vaccine for infectious bovine rhinotracheitis virusas claimed in claim 30, wherein said pharmaceutically effective amountis about 10⁴.5 to 10⁵.5 PFU.
 32. A method of immunizing an animalagainst infectious bovine rhinotracheitis comprising administering, toan animal, a pharmaceutically effective amount of an infectious bovinerhinotracheitis virus which fails to produce any functional TK as aresult of a deletion in the tk gene, produced by a processcomprising:(1) Constructing a hybrid plasmid comprising a cloning vectorand a DNA fragment of IBRV containing substantially all of the IBRV tkgene; (2) Co-transfecting, in tk⁺ host cells, the hybrid plasmid of step(1) with infectious DNA from a tk⁻ IBRV mutagen-induced mutant; (3)Selecting, in tk⁻ host cells, for tk⁺ IBRV from the virus produced instep (2); (4) Deleting DNA sequences from the hybrid plasmid of step (1)such that less than substantially all of the IBRV tk gene is present;(5) Co-transfecting, in tk⁺ host cells, IBRV tk⁺ DNA derived from thetk⁺ IBRV obtained in step (3) with the resulting tk⁻ hybrid plasmid ofstep (4); and (6) Selecting, in tk⁻ host cells, for tk⁻ IBRV from thevirus produced in step (5) so as to produce tk⁻ IBRV mutants which failto produce any functional TK as a result of a deletion in the tk gene.33. The method of immunizing an animal against infectious bovinerhinotracheitis as claimed in claim 32, wherein said deletion is about10 to 1500 bp in size.
 34. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 33, wherein 75 to750 bp in size.
 35. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 32, wherein thetk⁻ IBRV mutagen-induced mutant in step (2) is a temperature-resistantmutant such that the resulting mutant in step (6) is both temperatureresistant and a tk⁻ deletion mutant.
 36. The method of immunizing ananimal against infectious bovine rhinotracheitis as claimed in claim 32,additionally comprising step (7):(7) Propagating the resulting IBRVwhich fails to produce any functional TK as a result of a deletion inthe tk gene of step (6) at a nonpermissive temperature for atemperature-sensitive virus so as to select for and produce atemperature-resistant IBRV which fails to produce any functional TK as aresult of a deletion in the tk gene.
 37. The method of immunizing ananimal against infectious bovine rhinotracheitis as claimed in claim 32,wherein an oligonucleotide linker is present in the deletion site of theIBRV DNA of step (4) while retaining IBRV DNA sequences adjacent to eachside of the deletion site.
 38. The method of immunizing an animalagainst infectious bovine rhinotracheitis as claimed in claim 37,wherein said linker is about 7 to 40 bp in length.
 39. The method ofimmunizing an animal against infectious bovine rhinotracheitis asclaimed in claim 32, wherein said cloning vector is selected from thegroup consisting of pBR322, pMB9, pBR325, pKH47, pBR328, pHC79, phageCharon 28, pKB11, pKSV-10, pMAR420 and oligo (dG)-tailed pBR322.
 40. Themethod of immunizing an animal against infectious bovine rhinotracheitisas claimed in claim 39, wherein said cloning vector is pBR322.
 41. Themethod of immunizing an animal against infectious bovine rhinotracheitisas claimed in claim 32, wherein said hybrid plasmid of step (1) is pLATKdl NdeI.
 42. The method of immunizing an animal against infectiousbovine rhinotracheitis as claimed in claim 32, wherein in step (4) theIBRV DNA sequences adjacent to each side of the deletion are at least400 bp in size.
 43. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 32, wherein theresulting hybrid plasmid of step (4) is pLATK dl NdeI dl BgII/NG/SstI.44. The method of immunizing an animal against infectious bovinerhinotracheitis as claimed in claim 32, wherein said tk⁺ host cells areselected from the group consisting of RAB-9 (ATCC No. CRL-1414); primaryrabbit kidney cells, secondary rabbit kidney cells; rabbit cornea (SIRC)cells (ATCC No. CCL-60), rabbit kidney (LLC-RKl) cells (ATCC No.CCL-106), embryo bovine trachea (EBTR) cells (ATCC No. CCL-44), bovineturbinate (BT) cells (ATCC No. CRL-1390), and bovine kidney (MDBK) cells(ATCC No. CCL-22).
 45. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 44, wherein saidtk⁺ host cells are RAB-9.
 46. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 32, wherein thetk⁻ IBRV mutagen-induced mutant is selected from the group consisting ofthe non-temperature-resistant bromovinyldeoxyuridine-resistant IBRVmutant of IBRV(P8-2) and the temperature-resistant IBRV(B8-D53) (ATCCNo. VR-2066).
 47. The method of immunizing an animal against infectiousbovine rhinotracheitis as claimed in claim 46, wherein the tk⁻ IBRVmutagen-induced mutant is the temperature-resistant IBRV(B8-D53) (ATCCNo. VR-2066).
 48. The method of immunizing an animal against infectiousbovine rhinotracheitis as claimed in claim 32, wherein saidpharmaceutically effective amount is about 10⁴.5 to 10⁷ PFU.
 49. Themethod of immunizing an animal against infectious bovine rhinotracheitisas claimed in claim 48, wherein said pharmaceutically effective amountis about 10⁴.5 to 10⁵.5 PFU.
 50. The method of immunizing an animalagainst infectious bovine rhinotracheitis as claimed in claim 32,wherein said administering is conducted intranasally, intramuscularly,or subcutaneously.
 51. The method of immunizing an animal againstinfectious bovine rhinotracheitis as claimed in claim 32, wherein saidanimal is selected from the group consisting of bovine, swine, goats,and deer.
 52. The method of immunizing an animal against infectiousbovine rhinotracheitis as claimed in claim 51, wherein said animal isbovine.
 53. The method of immunizing an animal against infectious bovinerhinotracheitis as claimed in claim 32, wherein said virus has theidentifying characteristics of IBRV(NG) dl TK clone 1 (ATCC No.VR-2112).